Resistant Starch: Sources, Applications and Health … · Press The discovery of resistant starch represents one of the major developments in our understanding of the importance of

Press

The discovery of resistant starch represents one of the major developments in our understanding of the importance of carbohydrates for health in the past twenty years. There has been a steady increase in knowledge of its sources, uses and physiological effects, but more information is needed on the measurement and complex physiological functions of the various types. Resistant starch is now being incorporated into commercial foods as an ingredient to increase dietary fibre intake. Both commercial and natural sources of resistant starch have been linked to an array of health benefits, especially those related to gut health.

Resistant Starch: Sources, Applications and Health Benefits covers the intrinsic and extrinsic sources of resistant starch in foods, and compares different methods of measuring resistant starch, their strengths and limitations. Applications in different food categories are addressed by recognized academic researchers and industry experts. The book includes descriptions of how resistant starch performs in bakery, dairy, snack, breakfast cereals, pasta, noodles, confectionery, meat, processed food and beverage products. It also looks at the mechanism for improving intestinal health by resistant starch in comparison to prebiotic oligosaccharides and regular dietary fibres. Other chapters cover the impact of resistant starch on blood glucose response, satiety and gut microbiota composition, as well as metabolism in animal models and individual human subjects, and the book reviews research conducted into the ways in which resistant starch can support the prevention of colon cancer. Resistant Starch: Sources, Applications and Health Benefits is unique in focusing on this versatile and important ingredient, which will be of great use to a wide range of food professionals, including food scientists, product developers and manufacturers.

About the editors

Yong-Cheng Shi is Associate Professor and Director, Carbohydrate Polymers - Technology and Product Innovation, Department of Grain Science and Industry, Kansas State University, USA.

Clodualdo C. Maningat is Vice President, Applications Technology and Technical Services, MGP Ingredients, Inc., USA; Department of Grain Science and Industry, Kansas State University, USA.

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Resistant Starch

Shi and Maningat

Resistant Starch Sources, Applications and Health Benefits

Resistant Starch Sources, Applications and Health Benefits

Yong-Cheng Shi and Clodualdo C. Maningat

EDITORS

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Resistant Starch

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Resistant StarchSources, Applications andHealth Benefits

Edited by

Yong-Cheng Shi

Department of Grain Science and Industry, Kansas State University, USA

Clodualdo C. Maningat

MGP Ingredients, Inc., USA; Department of Grain Science and Industry,

Kansas State University, USA

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This edition first published 2013 # 2013 by John Wiley & Sons, Ltd.

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Resistant starch : sources, applications and health benefits / edited by Clodualdo C. Maningat,Yong-Cheng Shi.

pages cmIncludes bibliographical references and index.ISBN 978-0-8138-0951-9 (cloth)

1. Low-carbohydrate diet. 2. Starch–Health aspects. 3. Reducing diets. I. Maningat, Clodualdo C.,editor of compilation. II. Shi, Yong-Cheng, editor of compilation.

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Titles in the IFT Press series

� Accelerating New Food Product Design and Development (Jacqueline H. Beckley, Elizabeth

J. Topp, M. Michele Foley, J.C. Huang, and Witoon Prinyawiwatkul)

� Advances in Dairy Ingredients (Geoffrey W. Smithers and Mary Ann Augustin)

� Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals (Yoshinori Mine,

Eunice Li - Chan, and Bo Jiang)

� Biofilms in the Food Environment (Hans P. Blaschek, Hua H. Wang, and Meredith E. Agle)

� Calorimetry in Food Processing: Analysis and Design of Food Systems (G€on€ul KaletunSc)� Coffee: Emerging Health Effects and Disease Prevention (YiFang Chu)

� Food Carbohydrate Chemistry (Ronald E. Wrolstad)

� Food Ingredients for the Global Market (Yao-Wen Huang and Claire L. Kruger)

� Food Irradiation Research and Technology, Second Edition (Christoper H. Sommers and

Xuetong Fan)

� Foodborne Pathogens in the Food Processing Environment: Sources, Detection and Control

(Sadhana Ravishankar, Vijay K. Juneja, and Divya Jaroni)

� High Pressure Processing of Foods (Christopher J. Doona and Florence E. Feeherry)

� Hydrocolloids in Food Processing (Thomas R. Laaman)

� Improving Import Food Safety (Wayne C. Ellefson, Lorna Zach, and Darryl Sullivan)

� Innovative Food Processing Technologies: Advances in Multiphysics Simulation (Kai

Knoerzer, Pablo Juliano, Peter Roupas, and Cornelis Versteeg)

� Microbial Safety of Fresh Produce (Xuetong Fan, Brendan A. Niemira, Christopher J. Doona,

Florence E. Feeherry, and Robert B. Gravani)

� Microbiology and Technology of Fermented Foods (Robert W. Hutkins)

� Multiphysics Simulation of Emerging Food Processing Technologies (Kai Knoerzer, Pablo

Juliano, Peter Roupas and Cornelis Versteeg)

� Multivariate and Probabilistic Analyses of Sensory Science Problems (Jean-FranScoisMeullenet, Rui Xiong, and Christopher J. Findlay

� Nanoscience and Nanotechnology in Food Systems (Hongda Chen)

� Natural Food Flavors and Colorants (Mathew Attokaran)

� Nondestructive Testing of Food Quality (Joseph Irudayaraj and Christoph Reh)

� Nondigestible Carbohydrates and Digestive Health (Teresa M. Paeschke and William R.

Aimutis)

� Nonthermal Processing Technologies for Food (Howard Q. Zhang, Gustavo V. Barbosa-

C�anovas, V.M. Balasubramaniam, C. Patrick Dunne, Daniel F. Farkas, and James T.C. Yuan)

� Nutraceuticals, Glycemic Health and Type 2 Diabetes (Vijai K. Pasupuleti and James W.

Anderson)

� Organic Meat Production and Processing (Steven C. Ricke, Ellen J. Van Loo, Michael G.

Johnson, and Corliss A. O’Bryan)

� Packaging for Nonthermal Processing of Food (Jung H. Han)

� Practical Ethics for the Food Professional: Ethics in Research, Education and the Work-

place (J. Peter Clark and Christopher Ritson)

3GFFIRS 08/28/2013 13:43:24 Page 6

� Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions (Ross

C. Beier, Suresh D. Pillai, and Timothy D. Phillips, Editors; Richard L. Ziprin, Associate

Editor)

� Processing and Nutrition of Fats and Oils (Ernesto M. Hernandez and Afaf Kamal-Eldin)

� Processing Organic Foods for the Global Market (Gwendolyn V. Wyard, Anne Plotto,

Jessica Walden, and Kathryn Schuett)

� Regulation of Functional Foods and Nutraceuticals: A Global Perspective (Clare M. Hasler)

� Resistant Starch: Sources, Applications and Health Benefits (Yong-Cheng Shi and Clodualdo

Maningat)

� Sensory and Consumer Research in Food Product Design and Development (Howard R.

Moskowitz, Jacqueline H. Beckley, and Anna V.A. Resurreccion)

� Sustainability in the Food Industry (Cheryl J. Baldwin)

� Thermal Processing of Foods: Control and Automation (K.P. Sandeep)

� Trait - Modified Oils in Foods (Frank T. Orthoefer and Gary R. List)

� Water Activity in Foods: Fundamentals and Applications (Gustavo V. Barbosa-C�anovas,Anthony J. Fontana Jr., Shelly J. Schmidt, and Theodore P. Labuza)

� Whey Processing, Functionality and Health Benefits (Charles I. Onwulata and Peter J. Huth)

3GFFIRS 08/28/2013 13:43:24 Page 7

To my wife Lei and my son Gary – YCS

To my wife Josie, my daughter Barbara and my sister Susan – CCM

3GFFIRS 08/28/2013 13:43:24 Page 8

3GFTOC 07/20/2013 11:21:12 Page 9

Contents

Preface xvii

About the Editors xix

List of Contributors xxi

Acknowledgements xxv

1 Starch Biosynthesis in Relation to Resistant Starch 1Geetika Ahuja, Sarita Jaiswal and Ravindra N. Chibbar

1.1 Introduction 11.1.1 Starch components 1

1.1.2 Resistant starch 2

1.2 Factors Affecting Starch Digestibility 3

1.3 Starch Biosynthesis 4

1.4 Starch Biosynthesis in Relation to RS 61.4.1 ADP-glucose pyrophosphorylase (AGPase) 6

1.4.2 Starch synthases (SS) 6

1.4.3 Starch branching enzymes (SBE) 11

1.4.4 Starch debranching enzymes (DBE) 13

1.5 Concluding Remarks 13

Acknowledgements 15

References 15

2 Type 2 Resistant Starch in High-Amylose Maize Starchand its Development 23Hongxin Jiang and Jay-lin Jane

2.1 Introduction 23

2.2 RS Formation in High-Amylose Maize Starch 28

2.3 RS Formation During Kernel Development 29

2.4 Elongated Starch Granules of High-Amylose

Maize Starch 312.4.1 Structures of elongated starch granules 31

ix

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2.4.2 Formation of elongated starch granules 33

2.4.3 Location of RS in the starch granule 35

2.5 Roles of High-Amylose Modifier (HAM) Gene in

Maize ae-Mutant 36

2.6 Conclusions 37

References 38

3 RS4-Type Resistant Starch: Chemistry, Functionalityand Health Benefits 43Clodualdo C. Maningat and Paul A. Seib

3.1 Introduction 43

3.2 Historical Account of Starch Indigestibility 44

3.3 Starch Modification Yielding Increased Resistance

to Enzyme Digestibility 473.3.1 Cross-linked RS4 starches 50

3.3.2 Substituted RS4 starches 54

3.3.3 Pyrodextrinized RS4 Starches 56

3.4 Physicochemical Properties Affecting Functionality 57

3.5 Physiological Responses and Health Benefits 60

3.6 Performance in Food and Beverage Products 65

3.7 Conclusions and Future Perspectives 68

References 68

4 Novel Applications of Amylose-Lipid Complex asResistant Starch Type 5 79Jovin Hasjim, Yongfeng Ai and Jay-lin Jane

4.1 Introduction 79

4.2 Enzyme Digestibility of Amylose-Lipid Complex 804.2.1 Effects of lipid structure on the enzyme resistance

of amylose-lipid complex 81

4.2.2 Effects of the crystalline structure on the enzyme

resistance of amylose-lipid complex 82

4.2.3 Effects of amylose-lipid complex on the enzyme

resistance of granular starch 82

4.3 Production of Resistant Granular Starch Through

Starch-Lipid Complex Formation 834.3.1 Effects of fatty-acid structure on the RS content 83

4.3.2 Effects of debranching on the RS content 85

4.4 Applications of the RS Type 5 86

4.5 Health Benefits of RS Type 5 874.5.1 Glycemic and insulinemic control 87

x Contents

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4.5.2 Colon cancer prevention 89

4.6 Conclusion 91

References 92

5 Digestion Resistant Carbohydrates 95Annette Evans

5.1 Introduction 95

5.2 Starch Digestion 95

5.3 Physical Structures of Starch 975.3.1 Starch helices 98

5.3.2 Crystalline structures 99

5.3.3 Starch granule structure 99

5.4 Resistant Starch due to Physical Structure 100

5.5 Molecular Structure of Starch 102

5.6 Enzyme Resistance due to Molecular Structure 103

5.7 Conclusion 106

References 106

6 Slowly Digestible Starch and Health Benefits 111Genyi Zhang and Bruce R. Hamaker

6.1 Introduction 111

6.2 SDS and Potential Beneficial Health Effects 1126.2.1 Potential health benefit of SDS relative to RDS 113

6.3 The Process of Starch Digestion 1156.3.1 Enzyme action 115

6.4 Structural and Physiological Fundamentals of SDS 1166.4.1 Physical or food matrix structures related to SDS 117

6.4.2 Starch chemical structures leading to SDS 118

6.4.3 Other food factors that decrease digestion rate 120

6.4.4 Physiological control of food motility 121

6.5 Application-Oriented Strategies to Make SDS 1216.5.1 Starch-based ingredients 121

6.5.2 SDS generation in a food matrix 122

6.6 Considerations 123

References 123

7 Measurement of Resistant Starch and Incorporationof Resistant Starch into Dietary Fibre Measurements 131Barry V. McCleary


7.2 Development of AOAC Official Method 2002.02 133

Contents xi

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7.3 Development of an Integrated Procedure for the

Measurement of Total Dietary Fibre 136

References 142

8 In Vitro Enzymatic Testing Method and DigestionMechanism of Cross-linked Wheat Starch 145Radhiah Shukri, Paul A. Seib, Clodualdo C. Maningat,

and Yong-Cheng Shi


8.2 Materials and Methods 1488.2.1 Materials 148

8.2.2 General methods 148

8.2.3 Conversion of CL wheat starch to phosphodextrins

and 31PNMR spectra of the phosphodextrins 148

8.2.4 Digestibility of CL wheat starch 149

8.2.5 Thermal properties 150

8.2.6 Microscopic observation 150

8.2.7 Scanning electron microscope (SEM) 150

8.2.8 Statistical analysis 150

8.3 Results and Discussion 1518.3.1 Effects of a-amylase/amyloglucosidase digestion

on P content and chemical forms of the

phosphate esters on starch 151

8.3.2 Thermal properties 152

8.3.3 Starch granular morphology before and

after enzyme digestion 153

8.3.4 Digestibility 160

8.4 Conclusions 162

8.5 Acknowledgements 163

8.6 Abbreviations Used in This Chapter 163

References 163

9 Biscuit Baking and Extruded Snack Applicationsof Type III Resistant Starch 167Lynn Haynes, Jeanny Zimeri and Vijay Arora


9.2 Thermal Characteristics of Heat-Shear Stable

Resistant Starch Type III Ingredient 168

9.3 Application to Biscuit Baking: Cookies 172

9.4 Cracker Baking 175

9.5 Extruded Cereal Application 1789.5.1 Preparation of extruded RTE cereal and analysis 179

References 189

xii Contents

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10 Role of Carbohydrates in the Prevention of Type 2 Diabetes 191Thomas M.S. Wolever


10.2 Background 19110.2.1 Definition of diabetes 191

10.2.2 Types of diabetes 192

10.2.3 Complications of diabetes 192

10.2.4 Prevalence of diabetes 192

10.2.5 Risk factors for type 2 diabetes 193

10.3 Carbohydrates and Risk of Type 2 Diabetes 19310.3.1 Markers of carbohydrate quality 193

10.4 Pathogenesis of Type 2 Diabetes 195

10.5 Effect of Altering Source or Amount of Dietary

Carbohydrate on Insulin Sensitivity, Insulin Secretion

and Disposition Index 197

10.6 Mechanisms by Which Low-GI Foods Improve Beta-Cell

Function 19910.6.1 Glucose toxicity 199

10.6.2 Reduced serum free fatty acids (FFA) 200

10.6.3 Increased GLP-1 secretion 201

10.7 Conclusions 202

References 202

11 Resistant Starch on Glycemia and Satiety in Humans 207Mark D. Haub


11.2 Diet and Resistant Starch 208

11.3 Resistant Starch and Insulin Sensitivity 209

11.4 Current Theoretical Mechanism 209

11.5 Satiety 211

11.6 Fermentation and Gut Microbiota 212

11.7 Effect of RS Type 212

11.8 Summary 213

References 213

12 The Acute Effects of Resistant Starch on Appetiteand Satiety 215Caroline L. Bodinham and M. Denise Robertson

12.1 Appetite Regulation 215

12.2 Measurement of Appetite in Humans 216

12.3 Proposed Mechanisms for an Effect of Resistant Starch

on Appetite 217

Contents xiii

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12.4 Rodent Data 218

12.5 Human Data 221

References 225

13 Metabolic Effects of Resistant Starch 229Martine Champ

13.1 Fermentation of RS and Its Impact on Colonic

Metabolism 230

13.2 Resistant Starch, Glycemia, Insulinaemia and Glucose

Tolerance 235

13.3 RS Consumption and Lipid Metabolism 236

13.4 RS Consumption, GIP, GLP-1 and PYY Secretion 238

13.5 RS Consumption, Satiety and Satiation and

Fat Deposition 239

13.6 Conclusion 242

References 244

14 The Microbiology of Resistant Starch Fermentation in theHuman Large Intestine: A Host of Unanswered Questions 251Harry J. Flint


14.2 Identifying the Major Degraders of Resistant Starch

in the Human GI Tract 25214.2.1 The human colonic microbiota 252

14.2.2 Cultural studies 252

14.2.3 16S rRNA-based studies 253

14.3 Systems for Starch Utilization in Gut Bacteria 25414.3.1 Bacteroides spp. 255

14.3.2 Bifidobacterium spp. 255

14.3.3 Lachnospiraceae - Roseburia spp., Eubacterium

rectale and relatives 256

14.3.4 Ruminococcaceae 256

14.4 Metagenomics 256

14.5 Factors Influencing Competition for Starch as a Growth

Substrate 257

14.6 Metabolite Cross-Feeding 258

14.7 Impact of Dietary Resistant Starch upon Colonic Bacteria

and Bacterial Metabolites in Humans 259

14.8 Conclusions and Future Prospects 260

Acknowledgements 262

References 262

xiv Contents

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15 Colon Health and Resistant Starch: Human Studiesand Animal Models 267Suzanne Hendrich, Diane F. Birt, Li Li and Yinsheng Zhao

15.1 RS Classification 267

15.2 RS and Colon Health: Overview 267

15.3 RS, Gut Microbes and Microbial Fermentation 26815.3.1 RS and laxation 269

15.3.2 RS, IBS and diverticulosis 270

15.3.3 RS and IBD 270

15.3.4 RS and colon cancer risk – human studies 271

15.4 Colon Cancer Prevention – Animal Models 272

15.5 Conclusions 275

References 275

Index 279

Contents xv

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3GFPREF 07/20/2013 11:29:26 Page 17

Preface

Since the term ‘dietary fibre’ was first coined in 1953, it has undergone

several transformations with respect to its definition, composition, analytical

methodology and physiological effects. Its heterogeneous composition of

naturally-occurring non-starch polysaccharides, lignin and associated substan-

ces has grown to include other synthetic or novel fibres, comprising digestion-

resistant dextrins and resistant starches. Because of this diverse composition,

analysts are often confronted with the challenge of accurately quantifying the

level of total dietary fibre of food or beverage products. Dietary fibre is now

less frequently associated with bulk or regularity and is discussed much more

conspicuously with its role in attenuation of glycemic/insulinemic responses,

blood cholesterol lowering, satiety effects, weight management, large bowel

fermentation and changes in gut microbiota composition and metabolism in

regard to their impact on the general health and well-being of consumers.

Consumer demand for fibre-rich foods and beverages in the United States,

Europe and Asia-Pacific is rising due primarily to the preponderance of

positive epidemiological and scientific data and also an increase in consumer

awareness and support from dieticians and nutritionists. Ironically, however,

many Americans on average consume only about 50–60% of their recom-

mended daily intake of 25 g of fibre.

Resistant starch (RS), in particular, has captivated leading research scien-

tists and prominent educators, and their investigations have been featured

prominently in scientific literature on fibre. Many research activities on RS

highlighted its structure, composition, functionality, in vitro and in vivo

studies and performance in food and beverage products. RS has five types

or classes and, therefore, it provides diverse materials for research investiga-

tors. These, together with the commercial significance of RS, account for the

abundance of published articles and inventions in the scientific and patent

literature. Commercial sources of RS number around 30 – a substantial

increase since the first RS product was introduced to the market in 1993.

xvii

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The idea of writing this book was developed from the Carbohydrate

Division Symposium on resistant starch and health during the 2009 IFT

Annual Meeting in Anaheim, California. The symposium attracted speakers

who are leading researchers and scientists from the academia and the food

industry. In order to capture the important developments in RS, with

emphasis on sources, applications and health benefits, the editors embarked

on a project to write this book using the symposium papers plus the

contribution of invited scientists and academic professionals who excel

in this important area of RS.

There are 15 chapters in the book, covering various topics on RS, such as

its biosynthesis, types or classes, slowly digestible starch, methodology for

measurement and food applications, and also the physiological effects of

RS, primarily in the area of glycemic/insulinemic control, appetite/satiety,

gut microbiota metabolism and large bowel health. This book caters to a

wide audience and can be a valuable resource for students, professors,

research scientists, product developers and other food industry professio-

nals, as they investigate the ever-growing area of RS and its diverse

properties, numerous food and beverage applications, commercial signifi-

cance and physiological effects.

xviii Preface

3GFABOUT 07/20/2013 13:23:15 Page 19

About the Editors

Yong-Cheng Shi, Ph.D. is Professor and Director of the Carbohydrate

Polymers – Technology and Product Innovation group in the Department

of Grain Science and Industry at Kansas State University in Manhattan,

Kansas. He has authored or co-authored more than 40 journal articles and

book chapters and holds more than 15 patents. His research interests include:

structure and properties of starches; physical, chemical, and enzymatic

modifications of starches, biopolymers and flours; carbohydrate and health;

starch digestibility, resistant starch and dietary fibre; ingredient functionality

in cereal products; and developing technologies and products for food,

nutrition, emulsion, encapsulation, pharmaceutical and other industrial

applications.

Dr. Shi received his B.S. in Chemical Engineering from Zhejiang Univer-

sity (Hangzhou, China) and his M.S. and Ph.D. in Grain Science from Kansas

State University (Manhattan, Kansas). He is a professional member of the

American Association of Cereal Chemists International and Institute of Food

Technologists. He is an associate editor forCereal Chemistry and amember of

Advisory Board for Starch and Food Digestion journals.

Clodualdo ‘Ody’ C. Maningat, Ph.D. is Vice President of Applications

Technology and Technical Services at MGP Ingredients, Inc. in Atchison,

Kansas and Adjunct Faculty Member in the Department of Grain Science and

Industry at Kansas State University in Manhattan, Kansas. He is a member and

former chair of the Advisory Board of the Food Processing Center of the

University of Nebraska in Lincoln, Nebraska. He has authored or co-authored

more than 25 journal articles and book chapters in grain and food science

publications and holds more than 30 patents on grain-based technologies. His

research and business interests include: chemistry, modification and function-

ality of starches and proteins; analysis and function of dietary fibres; value-

addition concepts; technology of RS4-type resistant starch; physiological

xix

3GFABOUT 07/20/2013 13:23:15 Page 20

benefits of grain-derived ingredients; and research alliances with scientists and

product developers in the food industry, government and academia.

Dr. Maningat received his B.S. in Chemistry from Adamson University

(Manila, Philippines), his M.S. in Agricultural Chemistry from the University

of the Philippines at Los Banos (Laguna, Philippines) and his Ph.D. in Grain

Science from Kansas State University (Manhattan, Kansas). He is a profes-

sional member of the American Association of Cereal Chemists International,

Institute of Food Technologists, American Society of Baking and American

Chemical Society.

xx About the Editors

3GFCONT 07/20/2013 14:20:4 Page 21

List of Contributors

Geetika AhujaDepartment of Plant Sciences

College of Agriculture &

Bioresources

University of Saskatchewan

Canada

Yongfeng AiDepartment of Food Science and

Human Nutrition

Iowa State University

USA

Vijay AroraIngredient and Process Research

Mondelez International

USA

Diane F. BirtInterdepartmental Graduate Program

in Genetics

Department of Food Science and

Human Nutrition

Nutrition and Wellness Research

Center


USA

Caroline L. BodinhamDepartment of Nutritional Sciences

Faculty of Health and Medical

Sciences

University of Surrey

UK

Martine ChampINRA, UMR 1280

Physiologie des Adaptations

Nutritionnelles

Universite de Nantes, CRNH,

IMAD, CHU de Nantes, Nantes

France

Ravindra N. ChibbarDepartment of Plant Sciences


Bioresources


Canada

Annette EvansInnovation and Commercial

Development

Tate & Lyle

USA

xxi

3GFCONT 07/20/2013 14:20:4 Page 22

Harry J. FlintMicrobial Ecology Group

Rowett Institute of Nutrition and

Health

University of Aberdeen

Aberdeen, UK

Bruce R. HamakerWhistler Center for Carbohydrate

Research and Department of Food

Science

Purdue University

USA

Jovin HasjimQueensland Alliance for Agriculture

and Food Innovation

Centre for Nutrition and Food

Sciences

The University of Queensland

Australia

Mark D. HaubDepartment of Human Nutrition

Kansas State University

USA

Lynn HaynesIngredient and Process Research


USA

Suzanne HendrichInterdepartmental Graduate Program

in Genetics


Human Nutrition


Center


USA

Sarita JaiswalDepartment of Plant Sciences


Bioresources


Canada

Jay-lin JaneDepartment of Food Science and

Human Nutrition


USA

Hongxin JiangDepartment of Food Science and

Human Nutrition


USA

Li LiInterdepartmental Graduate Program

in Genetics


Human Nutrition


Center


USA

Clodualdo C. ManingatMGP Ingredients

Inc., USA; Department of Grain

Science and Industry


USA

Barry V. McClearyMegazyme International

Bray Business Park

Ireland

xxii List of Contributors

3GFCONT 07/20/2013 14:20:5 Page 23

M. Denise RobertsonDepartment of Nutritional

Sciences

Faculty of Health and Medical

Sciences

University of Surrey

UK

Paul A. SeibDepartment of Grain Science and

Industry


USA

Yong-Cheng ShiCarbohydrate Polymers –

Technology and Product

Innovation

Department of Grain Science and

Industry


USA

Radhiah ShukriDepartment of Grain Science and

Industry


USA

Thomas M.S. WoleverDepartment of Nutritional Sciences

University of Toronto

Canada; Division of Endocrinology

and Metabolism

St. Michael’s Hospital

Canada

Genyi ZhangSchool of Food Science and

Technology

Jiangnan University

China

Yinsheng ZhaoInterdepartmental Graduate Program

in Genetics


Human Nutrition


Center


USA

Jeanny ZimeriIngredient and Process Research


USA

List of Contributors xxiii

3GFCONT 07/20/2013 14:20:5 Page 24

3GFACKNOW 07/20/2013 13:31:42 Page 25

Acknowledgements

We are profoundly grateful to the chapter authors for their expertise and their

valuable contributions to make this book a reality. This is a tribute to their hard

work and the countless hours devoted in writing the chapters. A number of

scientists and academicians, to whom we extend sincere thanks, volunteered

their time to review and provide critique to the book’s contents. They are as

follows: Mike Gidley (University of Queensland), Ya-Jane Wang (University

of Arkansas), David Robbins (University of Kansas Medical Center), Jens

Walter (University of Nebraska, Lincoln), M. Denise Robertson (University of

Surrey), Paul A. Seib (Kansas State University), Steve Pickman (Consultant)

and Annette Evans (Tate & Lyle). The patience, accommodating attitude

and excellent editorial assistance of Mr. David McDade, Ms. Becky Ayre,

Mr. Sharib Asrar, Ms. Jasmine Chang and other Wiley staff are also gratefully

acknowledged.

xxv

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3GCH01 07/19/2013 16:58:7 Page 1

1 Starch Biosynthesis in Relationto Resistant Starch

Geetika Ahuja, Sarita Jaiswal andRavindra N. ChibbarDepartment of Plant Sciences, College of Agriculture & Bioresources,University of Saskatchewan, Canada

1.1 INTRODUCTION

1.1.1 Starch components

Starch is present in amyloplasts as semi-crystalline intracellular water-

insoluble granules, with alternating crystalline and amorphous layers. Starch

is a glucan homopolymer composed of one-quarter amylose (molecular mass

105–106Da) and three-quarters amylopectin (molecular mass 107–109Da),

along with traces of lipids (0.1–1.0%) and proteins (0.05–0.5%). Amylose is

essentially a linear glucan polymer, composed of a-1,4 linked glucose

residues with a degree of polymerization (dp) ranging between 800 (in maize

and wheat) to more than 4500 (in potato) with sparse branching

(approximately one branch per 1000 residues) (Morrison & Karkalas,

1990; Alexander, 1995). Structural and functional aspects of these glucan

polymers affect starch functionality and its end use.

Amylose chains are capable of forming single or double helices. On

the basis of orientation of its fibres in X-ray diffraction studies, amylose

can be divided into A- and B-type allomorphs (Galliard et al., 1987). In B-type

allomorph, six double helices are packed in an anti-parallel hexagonal

mode surrounding the central water channel (36 H2O per unit cell). In

A-type, the central water channel is replaced by another double helix, making

the structure more compact. In this allomorph, only eight molecules of water

per unit cell are inserted between the double helices (Galliard et al., 1987).

Amylopectin is a highly branched glucan polymer, in which a-1,4 linked

glucose residues are interspersed with a-1,6-glucosidic linkages (4–5%)

1

Resistant Starch: Sources, Applications and Health Benefits, First Edition.

Edited by Yong-Cheng Shi and Clodualdo C. Maningat.

� 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

3GCH01 07/19/2013 16:58:7 Page 2

which introduce branches, and a degree of polymerization ranging from

105–107 glucose units (Myers et al., 2000). Chain lengths of 20–25 glucose

units between branch points are typical. The branches in the amylopectin

molecule are arranged in clusters (Bul�eon et al., 1998).

An amylopectin molecule typically consists of three types of chains, which

are either located within a single cluster or connect two or more clusters

(Hizukuri et al., 1986; Thompson et al., 2000). In amylopectin, only theC-chain

has a reducing end oriented towards the centre or hilum of the granule. Attached

to the C-chain with a-1,6 linkages are the B-chains. These can support other

B- or A-chains. The A-chains are the outermost chains, which do not support

any other chains. A- and B-chains form clusters and B-chains can span and

supportmultiple clusters.A-chains typically consist of 6–12 glucosemolecules,

while B-chains may contain 13–24 or up to 50 or more glucose molecules,

depending on the number of clusters they span. In the section which does not

contain a-1,6 branch points, two neighbouring glucose chains form a double

helix, and these double helices form a crystalline pattern. All of these structures

are attached by hydrogen bonds. The sections where the branch points of the

amylopectin are located are amorphous and contain amylose molecules.

1.1.2 Resistant starch

More than 50% of calorific requirement of human diet is fulfilled by starch-

based foods, and the quality and quantity of starch-based food affect overall

blood glucose and homeostasis in humans. Starch digestion in humans is

initialized by salivary a-amylases in the oral cavity, followed by pancreatic

a-amylase and the intestinal brush border glucoamylases, maltase-glucoa-

mylase, and sucrase-isomaltase (Nichols et al., 2003). Brush-border enzymes

convert the resultant products of digestive process into maltase-glucoamylase

and sucrase-isomaltase, which enter the vascular system (Lehmann & Robin,

2007). Based on its in vitro enzymatic hydrolysis, the rate of glucose release

and its absorption in the gastrointestinal tract, starch is classified as either

readily digestible starch (RDS), slowly digestible starch (SDS) or resistant

starch (RS) (Englyst et al., 1992).

According to Englyst et al. (1992), based on in vitro kinetic assay, RDS is

broken down into glucose molecules in �20 minutes, while SDS is the

fraction which gets digested in �100 minutes. Both RDS and SDS are

completely digested in the small intestine. RS is referred to that portion of

starch which is not hydrolyzed until about 120 minutes have elapsed. It passes

through the small intestine undigested, but is fermented in the large intestine

by gut microflora. Physiological benefits of RS include hypoglycaemic effects

and production of short chain fatty acids (SCFA) particularly butyrate, which

2 Resistant Starch

3GCH01 07/19/2013 16:58:7 Page 3

has been reported to lower lumen pH, making it a less conducive environment

for cancer and other diseases (Topping & Clifton, 2001; Wei et al., 2010).

A medium-to-high amount of SDS has been reported for native normal

maize starch (Axelsen et al., 1999), waxy starches (Weurding et al., 2001),

millet and sorghum (Benmoussa et al., 2006) and legumes (Hoover & Zhou,

2003). A few researchers have reported a higher rate of digestibility for cereal

starches than tuber starches such as potato (Fannon et al., 1992; Benmoussa

et al., 2006). On the basis of its botanical source, physical or chemical

processing, RS can be divided into four types. RS1 is physically inaccessible

due to its location in the food, RS2 escapes digestion because of its granular

structure, RS3 is retrograded starch and RS4 is chemically modified starch

(Brown, 2004).

1.2 FACTORS AFFECTING STARCH DIGESTIBILITY

Starch enzymatic hydrolysis and RS are influenced by several factors, both

extrinsic and intrinsic properties of starch granules. Extrinsic factors, which

include starch granule surface characteristics such as porosity of granule and

pit formation between the surface and centre of the granule (Fannon et al.,

1992), or exo-corrosion (Gallant et al., 1997), affect starch digestibility.

Intrinsic properties of starch granules, such as packing of amorphous and

crystalline regions (Gallant et al., 1992; Zhang et al., 2006), or interaction of

amylose with other components such as lipids (Crowe et al., 2000), proteins

(Escarpa et al., 1997) and/or enzyme inhibitors (Bjorck et al., 1987), also

influence starch digestibility. Reduced digestibility of tuber starch granules

has been attributed to their large and smooth surface, along with their surface

properties.

The amylose to amylopectin ratio is an important determinant of starch

digestibility. Amylose and amylopectin have different structural and physio-

logical characteristics and, hence, exhibit different reactions within the body

during digestion and subsequent release of glucose molecules for absorption.

The amylose to amylopectin ratio is a major determinant for RS2 and RS3

(Sajilata et al., 2006).

A positive correlation exists between amylose concentration and RS

formation (Ito et al., 1999). The straight chains of amylose limit the access

of small intestine b-amylases to the two terminal glucose units on the

amylose chain (besides, two terminal ends may not be accessible due to

folding of a polymer). In contrast, the highly branched structure of amylo-

pectin provides multiple terminal end glucose units that b-amylases can

access readily.

Starch Biosynthesis in Relation to Resistant Starch 3

3GCH01 07/19/2013 16:58:7 Page 4

During cooking, starch is gelatinized and amylose molecules are leached

out of the swollen starch granules as coiled polymers which, on cooling,

associate as double helices and form hexagonal networks which resist

digestion. In waxy starches, instead of this network, aggregate formation

occurs, and this is more susceptible to hydrolysis by amylases.

The intensity of starch digestion is also affected by the degree of polymeri-

zation and/or branching of glucan polymers, i.e. a reduction in the rate of

hydrolysis with increased branching due to steric hindrance (Park & Rollings,

1994). Gamma irradiation-generated rice mutants high in RS showed

increased proportion of short chains with DP� 12, decreased proportion of

intermediate chains of 13�DP� 36 and decrease in long chains with

DP� 37 (Shu et al., 2007).

Another report, by Ao et al. (2007), mentions that b-amylase and malto-

genic a-amylase mediated partial reduction of outer branch chains of amylo-

pectin reduces overall starch digestion rate, which was related to an increase in

the amount of a-1,6 linkages and decrease in a-1,4 linkages. Changes in the

amylopectin chain length distributions facilitated retrogradation to produce

B- and V- type crystalline structures, leading to more resistant starch. It is

generally believed that increased proportion of longer chains makes the starch

more resistant to digestion. A possible reason could be that longer chains form

longer and more stable helices, which are further stabilized by hydrogen

bonds distributed over the entire crystalline region and cause decreased

digestibility (Lehmann & Robin, 2007).

1.3 STARCH BIOSYNTHESIS

Plants have a unique ability to capture light energy and to fix carbon dioxide

and water to form triose sugars that act as precursor of simple and complex

carbohydrates. Photosynthesis in the plants’ chloroplast results in the produc-

tion of triose-phosphates, reducing equivalents and ATP. The triose phos-

phates are either transported by triose-phosphate transporters to the cytosol, or

are converted to phosphorylated compounds, including fructose-6-phosphate

in the plastid. During the light period, chloroplasts synthesize transitory starch

which, at night, is broken down into constituent sugars and transported to the

storage organs. In contrast, in amyloplasts, these precursors are used to

synthesize storage starch.

Analogous to chloroplasts in green tissues, storage organs contain amy-

loplasts which are albino plastids and devoid of internal membrane structure.

These specialized plastids act as processing and storage unit for starch in plant

cells. Fructose-6-phosphate in chloroplasts is used both for regeneration of

4 Resistant Starch

3GCH01 07/19/2013 16:58:7 Page 5

ribulose-1,5-bisphosphate and production of glucose-1-phosphate through

glucose-6-phosphate. Conversion of glucose-1-phosphate and ATP to

ADP-glucose by ADP-glucose pyrophosphorylase (AGPase) is the first

committed step in starch synthesis.

In addition to AGPase, other enzymes involved in the starch (especially

amylopectin) biosynthetic cascade include starch synthases (SS), starch branch-

ing enzymes (SBE) and debranching enzymes (DBE) (Smith et al., 2001; James

et al., 2003; Zeeman et al., 2010). Amylose is synthesized exclusively by

granule-bound starch synthase-I (GBSSI). The glucose moiety from ADP-

glucose is used to elongate an already existing glucan chain. Starch synthases

catalyze the formation of a-1,4 glucosidic linkage between the glucose units toform a linear chain. SS require a primer for elongation of glucose chain.

The initiation of glucan polymerization reaction is poorly understood. One

hypothesis suggests the presence of glycogenin-like self glycosylating protein

as primer for amylopectin synthesis and addition of D-glucose occurs to the

non-reducing end of a growing glucan chain (Chatterjee et al., 2005). Another

hypothesis is the de novo synthesis of glucan chains mediated by a two-site

insertion mechanism. Two glucose units from ADP-glucose complex with the

active site of starch synthase, and are subsequently added to the reducing end

of glucan chain (Mukerjea & Robyt, 2005).

Four starch synthase isoforms (SSI, SSII, SSIII, SSIV) play important role

in elongating different regions of amylopectin. Therefore, alterations in SS

activities would affect the amylopectin fine structure. Branches in amylopec-

tin and amylose are introduced by SBE, which catalyze the cleavage of an

a-1,4 linkage and join the cleaved chain to another glucan chain through a-1,6glucosidic linkage. Two classes of SBE (i.e. SBEI and SBEII) exist, which

have different substrate specificities.

Finally, debranching enzymes (isoamylase and pullulanase) act to trim the

outer branches of amylopectin molecule to form ordered branch structure and

packaging of the molecule into starch granules. Since multiple isoforms of

starch biosynthetic enzymes exist in the endosperm and have specific func-

tions, mutations in any of these genes would therefore lead to a change in

starch content, structure and functional properties.

In addition to the core enzymes, other enzymes, such as phosphorylases,

disproportionating enzymes and dikinases (glucan water dikinase, phos-

phoglucan water dikinase) also play important roles in starch metabolism.

Starch phosphorylation involves dikinases such as glucan water dikinase

(GWD, mol wt 155 kDa) and phosphoglucan water dikinase (PWD, mol wt

130 kDa), which phosphorylate the C6 and C3 positions of glucose units of

amylopectin, respectively – an important factor in starch degradation

(Fettke et al., 2009).


3GCH01 07/19/2013 16:58:7 Page 6

1.4 STARCH BIOSYNTHESIS IN RELATION TO RS

1.4.1 ADP-glucose pyrophosphorylase (AGPase)

AGPase catalyzes the synthesis of ADP-glucose from ATP and glucose-

1-phosphate. It is the first step in starch biosynthesis, and AGPase is also a key

regulatory enzyme in the starch biosynthetic pathway. AGPase consists of two

large and two small subunits, which affect allosteric and catalytic properties of

the enzyme. Allosteric regulation of this enzyme plays a critical role in

determining the amount of starch produced (Hannah & James, 2008). AGPase

is allosterically activated by 3-phosphoglyceric acid (3-PGA) and inhibited by

inorganic phosphate (Pi) in many plant tissues (Preiss et al., 1996). Genetic

and biochemical manipulation of its sensitivity towards Pi resulted in increase

in crop productivity (starch yield) due to increased sink strength (Wang et al.,

2007; Sakulsingharoj et al., 2004; Smidansky et al., 2002). AGPase activity is

also redox regulated (Hendriks et al., 2003).

In general, the active form of AGPase is present in the plastids of mature

cereal tissues and sink tissues of non-cereal plants. Developing cereals

however, differ, with most of their AGPase activity localized mainly in the

cytosol of endosperm cells. Specific transporters/ADP-glucose transporter

channels are involved in the trafficking of the resultant ADP-glucose. In non-

cereal plants, the sucrose to starch pathway comprises plastid import of

hexose phosphates, which can be used in other biosynthetic processes in

addition to starch synthesis. In contrast, in cereals, carbon entering the plastid

as ADP-glucose is committed to starch synthesis (James et al., 2003).

MutationsinAGPaseandADP-glucosetransportershavebeenshowntoaffect

the total starch content in maize, barley, pea and potato (Hylton et al., 1992;

Shannon et al., 1998; Tjaden et al., 1998; Patron et al., 2004). The maize

Shrunken-2 and Brittle-2 mutants have lesions in the large and small subunits

of the cytosolic AGPase, respectively (Hannah & Nelson, 1976). Shrunken-2

mutant kernels are deeply dented, with floury endosperm that has 25% reduced

starch but is sweet due to high sucrose concentration (Hutchinson, 1921).

Similarly Brittle-2 mutant kernel germinates poorly, is dark and shrunken and

has25–34% lower starch thannormal (Preiss et al., 1990).Abarleymutant,Risø

16, isassociatedwithadeletioninthesmall subunitofcytosolicAGPaseresulting

in reduced starch concentration and seed weight (Johnson et al., 2003). These

changes in starch concentration have not been associated to RS (Table 1.1).

1.4.2 Starch synthases (SS)

Starch synthases catalyze the transfer of glucose unit from ADP-glucose to

non-reducing end of an already existing glucan chain, thus forming a-1,4

6 Resistant Starch

3GCH01 07/19/2013 16:58:9 Page 8

linkage. Cereal endosperms contain at least five SS classes, based on their

conserved primary amino acid sequences. SSI and SSII are present mostly in

the stroma (Fujita et al., 2006), whereas SSIII and SSIVare present both in the

stroma and starch granule (Denyer et al., 1995; Dai, 2010) and are primarily

involved in amylopectin synthesis. GBSSI is bound to starch granules and is

required for amylose synthesis. Recently, GBSSI has also been shown to

participate in the elongation of amylopectin chains, particularly for very long

branches (Yoo & Jane, 2002). The chain elongation pattern differs for each

isoform and varies with plant species (Smith et al., 1997). In addition to their

specialized functions, some SS overlap in their functional role, while others

are unique (Rold�an et al., 2007).

1.4.2.1 Granule bound starch synthase-I

GBSSI (also known as waxy protein) present in the interior of starch granule is

essential for amylose synthesis. Plants lacking GBSSI enzymatic activity

produce starch without amylose, which is also called waxy starch. In wheat,

GBSS has two isoforms, GBSSI and GBSSII (Nakamura et al., 1998; Vrinten

& Nakamura, 2000). Another isoform, GBSSIb, exclusive to the pericarp

region, has been reported in barley (James et al., 2003). This is involved in

transient starch accumulation, which enhances the sink strength of the young

caryopsis (Patron et al., 2002).

In vitro study using ADP[14C] glucose as precursor of starch biosynthesis

in isolated starch granules showed uptake of malto-oligosaccharides of DP

2–7 by GBSSI as primers for amylose synthesis (Denyer et al., 1996).

GBSSI is also reported to be involved in the elongation of long chains of

amylopectin (Yoo & Jane, 2002; Craig et al., 1998). GBSSI elongates the

glucan chains which are confined to the semi-crystalline region of the

granule and cannot form branches. Consequently, the chains remain linear

and are known as amylose, or long-branch chains of amylopectin (Jane

et al., 2010).

1.4.2.1.1 Amylose in relation to RS formation

Amylose contributes to the formation of RS2 and RS3. Deficiency of GBSS1

activity produces starchmade of only amylopectin (waxy starch). Rate of starch

digestibility is high inwaxy andpartiallywaxy starch (reducedRS) compared to

normal starch from several plants (Rooney & Plugfelder, 1986; Bertoft et al.,

2000; Li et al., 2004; Chung et al., 2006; Asare et al., 2011). In a recent study on

starch structure and in vitro enzymatic hydrolysis using barley atypical amylose

concentration starch (Table 1.1), Asare et al. (2011), using atomic force

microscopy, reported high poly-dispersity indices for normal (1.4) and

8 Resistant Starch

3GCH01 07/19/2013 16:58:9 Page 9

increased amylose starch genotype (1.25), compared to near (partially) waxy

starch genotypes (0.33). They also concluded that energy requirement for

gelatinization and hydrolysis of waxy starch is lower than for normal or

high-amylose starch.Waxy starches aremore susceptible to hydrolytic enzymes

compared to starch granules with significant amylose concentration.

Hu et al. (2004) investigated three types of rice cultivars with varying

amylose content for in vitro hydrolysis and glycemic index determination.

They concluded quicker, complete and significantly higher rates of starch

hydrolysis for waxy and low-amylose rice than for intermediate and high-

amylose rice. In a more practical approach for estimating RS contribution for

amylose, Hung et al. (2005) substituted high-amylose wheat flour for normal

wheat flour in bread-making and observed higher RS content in the substituted

bread. Physical increase in amylose content through retrogradation

and extended cooling after cooking can also lower digestibility (Blazek &

Copeland, 2010).

1.4.2.2 Starch synthase-I

In maize, SSI is responsible for extending shorter A and B1 chains up to a

critical chain length, making it unsuitable for its own catalysis (Commuri &

Keeling, 2001). In rice, retrotransposon Tos17 insertion mediated SSI-defi-

cient mutant lines showed starch phenotype with decreased amylopectin

chains of DP8–12, but increased chains of DP6–7 and 16–19. This suggests

that SSI functions in generating DP8–12 chains from shorter chains of DP6–7

emerging from the branch point of A and B1 chains (Fujita et al., 2006).

Amylose synthesis was not affected by this mutation, and its effect on starch

hydrolysis has not been reported.

1.4.2.3 Starch synthase-II

In cereal endosperm, SSII synthesizes intermediate-length branch chains of

amylopectin (see review by Jane et al., 2010). Yamamori et al. (2000)

produced triple null wheat line lacking starch granule protein-1 (SGP1),

identified as SSIIa and homologous to maize SSIIa (Li et al., 1999). Lack of

SGP1 showed amylopectin with increased short chains of DP 6–10, a decrease

in intermediate chains of DP 11–25 and a concomitant increase in apparent

amylose concentration (30.8–37.4%).

In a subsequent study (Yamamori et al., 2006), wheat lines lacking SGP1

showed an increase in resistant starch level (3.6%) compared to normal wheat

(0.02%). In a similar approach, wheat lines deficient in SSII A and B genome

polypeptides resulted in increased amylose (32%) starch, as determined by


3GCH01 07/19/2013 16:58:9 Page 10

HP-SEC analyses (Chibbar & Chakraborty 2005; Lan et al., 2008). SSIIa

deficient maize (sugary2 mutation due to insertion in SSIIa) genotypes

showed an increase in abundance of short (DP 6–11) and medium (DP

13–25) chains. This mutation also resulted in an increase in apparent amylose

concentration from 26–40% (Zhang et al., 2004). In rice, japonica type has a

higher short to long chains ratio than indica type but, contrary to wheat and

maize, indica rice has higher amylose concentration than japonica rice

(Umemoto et al., 1999, 2002).

In barley, sex6mutation on chromosome 7H due to G!A transition results

in an early stop codon, thus inhibiting C-terminal translation of the active site

of SSIIa (Morell et al., 2003). The major effect of SSIIa inactivity is an

increase in amylose concentration (65–70%) in the mutants, which increases

RS content. In addition, a change in starch crystallinity from A-type to a

mixture of B- and V-type was also reported. V-type crystallinity indicates the

formation of amylose-lipid complexes, which inhibit starch swelling, and it

resists digestion by amylolytic enzymes (Morell et al., 2003). A barley

cultivar, Himalaya-292, which has an inactive SSIIa, produces increased

amylose starch and higher RS content. This RS-rich diet when fed to rats

changes its bowel SCFA (Bird et al., 2004).

A similar pattern of change with the SSII mutation on amylopectin fine

structure and amylose content has been reported in potato (Edwards et al.,

1999) and pea (Craig et al., 1998). SSIIa mutation in pea rug5 decreases

intermediate length amylopectin chains (B2 and B3) and produces a higher

(�35%) amylose concentration starch Table (1.1) (Craig et al., 1998).

1.4.2.4 Starch synthase-III

Amylopectin long B-chains are synthesized by SSIII. Mutation in maize SSIII

is called dull-1 (du1), which has a starch phenotype of amylopectin with

decreased proportion of long B-chains, enriched short-branch chains and

moderately increased amylose content (Wang et al., 1993). SSIII mutation

also affects SSII and SBEIIa and is capable of altering endosperm starch

structure (Gao et al., 1998). Ryoo et al. (2007) reported a mutation in rice

SSIII OsSSIIIa (floury, flo), which produced small and round starch granules

and endosperm with a loosely packed central portion, exhibiting a floury-like

phenotype. In rice flo mutant lines, amylopectin chains with DP� 30 were

reduced, suggesting that OsSSIIIa has a role in the generation of relatively

longer chains of amylopectin (i.e. B2 and B3 to B4). Concomitantly, a 2–4%

increase in the ratio of amylose to amylopectin was also observed.

In addition to its role in extending glucan chains, SSIII influences

starch structure through its association with other starch metabolizing

10 Resistant Starch

3GCH01 07/19/2013 16:58:9 Page 11

enzymes. Arabidopsis SSIII mutants AtSSIII1 and AtSSIII2 showed

increased starch concentration compared to wild type, suggesting a nega-

tive regulatory role of SSIII in biosynthesis of transient starch (Zhang

et al., 2005). However, no report is available on the effect of SSIII

mutation on starch digestibility.

1.4.2.5 Starch synthase-IV

In rice, two SSIV genes, SSIVa and SSIVb, have been shown to be expressed

during grain filling, both in the pericarp and the endosperm (Hirose & Terao,

2004). Arabidopsis SSIV mutants show a reduction in leaf starch concentra-

tion (Rold�an et al., 2007) and a striking reduction in leaf starch granules,

which suggests a role for SSIV in starch granule initiation. Recently, it has

been shown in an in vitro assay that SSIV has high SS activity when malto-

triose is used as primer (Szydlowski et al., 2009). To date, no cereal plants

deficient in SSIV activity have been characterized.

1.4.3 Starch branching enzymes (SBE)

Starch branching enzymes cleave a-1,4 linkages and transfer a free reducing

C-1 to C-6 hydroxyl group of glucose-unit in another chain, forming a new

a-1,6 branch linkage. Since branching is an essential part of amylopectin

synthesis, it will therefore be dependent on the available concentration of

needed SBE.

Based on primary amino acids sequence similarity and substrate specific-

ity, two major types of SBE (SBEI and SBEII) have been identified in cereals.

In vitro studies in maize suggest that SBEI prefers amylose as substrate and

transfers longer chains, whereas SBEII uses amylopectin as substrate and

transfers shorter chains (Guan & Preiss, 1993). In wheat, SBEII is further

divided into two�85% similar isoforms, SBEIIa and SBEIIb, with apparently

similar molecular weight (Rahman et al., 2001). In addition to this, a larger

form of SBEI, SBEIc (152 kDa) has been reported in wheat (Ba�ga et al.,

2000), which is preferentially associated with large A-type granules (Peng

et al., 2000). In dicots like pea and potato, two isoforms of SBE viz. SBEI and

SBEII (or, SBE B and SBE A) have been reported (Burton et al., 1995;

Poulsen & Kreiberg, 1993).

In maize, mutation in SBEIIb resulting in high-amylose starch is known as

amylose-extender (ae) (Stinard et al., 1993). This results in cereal starch with

high-amylose concentration (>50%) and amylopectin with more long branch-

chains and fewer short branch-chains (Jane et al., 1999). Similarly, another

report suggested a higher proportion of long chains (DP� 38) and a marked


3GCH01 07/19/2013 16:58:9 Page 12

reduction in short chains of DP� 17 in ae rice endosperm (Nishi et al., 2001).

It also showed a significant increase in apparent amylose concentration from

25–35%.

The very long chains of ae mutant amylopectin develop B-type crystallin-

ity (Kasemsuwan et al., 1995; Hizukuri et al., 1983), which favour slow

enzymatic digestion. These results corroborated a similar study in maize (Li

et al., 2008), where aemutants showed significant increase in chain lengths of

amylopectin and higher apparent and absolute concentrations of amylose.

Further, the mutants also showed considerably higher RS content (39.4–

43.2%) compared to the parents (11.5–19.7%). A commercial product con-

taining�80% amylose, called Hi-maize, has been derived from this mutation.

Hi-maize has been added to wheat products to increase RS amount (Brown,

2004).

In a recent study, barley RNAi mediated inhibition of SBEIIa and SBEIIb

activity altered starch composition and structure (Regina et al., 2010). The

study revealed that a reduction in expression of both SBEIIaþ b to >80%

elevated the amylose content to >65% from 28% in wild type resulting in a

significant increase in RS content (Table 1.1). However, they observed minor

differences when either enzyme was down-regulated. Also, reduction in

expression of both SBEIIaþ b showed an increase in the proportion of chains

of DP<9 and DP>15 and a consequent decrease in the number of medium

chains (DP9–13).

A similar trend has previously been reported in wheat, where an increase in

amylose content (<70%) in SBEIIa mutants was observed by simultaneous

inhibition of expression of both the SBE II isoforms (Regina et al., 2006). In

addition, decrease in proportion of amylopectin chains of DP4–12 and an

increase in chains of DP> 12 was also seen. In vivo feeding studies in rats

using high-amylose wheat meal showed higher amount of RS and lower

glycemic index in comparison to wild type wheat diet (Regina et al., 2006). In

potato, inhibition of SBE A and SBE B resulted in a very high-amylose

phenotype (up to �89% by potentiometric determination), while normal high

molecular weight amylopectin was absent (Schwall et al., 2000). This type of

starch would have lower digestibility.

Yao et al. (2009) studied four corn types with different doses of amylose-

extender(ae) and floury-1(fl1) alleles in the endosperm. Amylose and RS

contents followed a similar pattern with highest values in aeaeaeae (amylose

¼ 58.3%; RS¼ 55.2%). They also observed higher proportion of longer

branch chains with DP� 25 in these mutants. Since amylose-extender

mutation reduces SBEIIb activity, it results in an increase in amylose to

amylopectin ratio, which in turn increases RS content.

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1.4.4 Starch debranching enzymes (DBE)

Final packaging of the starch granule requires the trimming of extra branches.

Debranching enzymes have been postulated to play this important role in

amylopectin biosynthesis (Ball et al., 1996; Myers et al., 2000; Nakamura

et al., 2002). Two different mechanisms for DBE mode of action have been

proposed. The ‘preamylopectin-trimming model’ suggests that the outer

branches of preamylopectin molecules are trimmed by DBE to facilitate

chain elongation by SS (Mouille et al., 1996; Myers et al., 2000). This will

form amylopectin with an ordered branch structure and allow packaging of the

molecule in starch granules. In addition, glucan chains released by DBE’s

action on amylopectin can be elongated by GBSSI to form the amylose

fraction.

According to the ‘soluble glucan recycling model’, DBE participates in

degradation of short chain glucan molecules produced either by SS or SBE

action to prevent accumulation of highly branched soluble polymers at the

expense of amylopectin formation (Zeeman et al., 1998; Smith, 2001).

Endosperms deficient in DBE activity by lesions in DBE genes result in

the formation of phytoglycogen instead of amylopectin from soluble glucans

(Zeeman et al., 1998).

Two major DBE classes are recognized: isoamylases, which trim packed

structures (like glycogen); and pullulanases, which act on more open struc-

tures (like pullulan). Three types of isoamylases have been identified in cereal

endosperm (Kubo et al., 2005) and in potato (Hussain et al., 2003). Lack of

isoamylase-1 in rice (sugary-1, su-1), and barley (isa-1) resulted in small but

significant alteration in amylopectin chain length distribution (Kubo et al.,

2005). In mutant lines, starch granules were shrunken, irregular and com-

pound (reviewed in James et al. (2003)).

Pullulanase type DBEmutation is termed ZPU1 in maize. ZPU1 is an endo-

acting enzyme that cleaves only very short branch chains and it is activated by

redox status and inhibited by high sugar (Dinges et al., 2003). A similar report

on wheat limit-dextrinase-type-DBE activity suggests its redox regulation

(Repellin et al., 2008). Mutations in debranching enzymes, however, have not

been reported to be associated with resistant starch (Table 1.1).

1.5 CONCLUDING REMARKS

Starch biosynthesis is a complex process in which starch biosynthetic

enzymes act in a coordinated manner to produce amylopectin, which is


3GCH01 07/19/2013 16:58:9 Page 14

architecturally conserved in starches from different botanical sources.

Genetic strategies, by identifying genotypes with lesion(s) in gene(s)

encoding starch biosynthetic enzymes, have revealed the role of each

enzyme or its isoform in the synthesis of amylose and amylopectin

constituent glucan chains and consequent alteration in starch composition

and amylopectin architecture.

It has also been found that mutations in one locus in starch biosynthetic

pathway affects one or more other starch biosynthetic enzymes. Maize ae

mutant has a lesion in SBEIIb gene, but SBEI activity is reduced or absent

and changes the properties of an isoamylase type DBE (Colleoni et al.,

2003). Conversely, genetic lesions in pullulanase (zpu-204) or isoamylase

(sul-si) type DBE reduce SBEIIa activity, although SBEIIa polypeptide is

not altered or reduced (James et al., 1995; Dinges et al., 2003). Lesions in

SSII genes which reduce SSII activity also reduce/eliminate the binding of

SSI, SBEIIa and SBEIIb within the granule matrix, although these enzymes

have not lost their affinity to amylopectin or starch (Morell et al., 2003;

Umemoto & Aoki, 2005). These observations suggest that key starch

biosynthetic enzymes form protein complexes (Tetlow et al., 2004). Using

isolated amyloplasts, starch biosynthetic enzyme complexes have been

shown in wheat and maize (Tetlow et al., 2004; Hennen-Bierwagen

et al., 2008).

In a recent proteomics study, it has been shown that phosphorylation of

GBSSI, SBEIIb and Pho 1 is needed for their incorporation in to starch

granules (Grimaud et al., 2008). The concept of starch biosynthetic enzymes

acting in a complex and its formation is dependent upon the phosphorylation

status of constituent enzymes and is an additional level of control in starch

biosynthesis.

There is significant interest in increasing amylose concentration in cereal

and tuber starches. Increased amylose concentrations have been attributed to

both SBE and SS isoforms. In addition to natural mutants in maize (ae) and

barley (sex6), amylose to amylopectin ratios in starch have been manipulated

by altering GBSSI and SBEII (waxy/amylose extender) activity in wheat

(Lafiandra et al., 2010; Sestili et al., 2010; Regina et al., 2006), in maize

(Jiang et al., 2010) and in rice (Wei et al., 2010). In wheat and barley, very

high amylose concentrations were obtained by RNAi mediated inhibition of

Sbe2a and Sbe2b genes (Regina et al., 2006, 2010). Recent advances in

understanding starch biosynthesis, combined with innovations in genomics

(Ganeshan et al., 2010), can be used to develop cereal genotypes with

increased amylose concentrations and alteration in amylopectin architecture

which can be used to produce RS.

14 Resistant Starch

3GCH01 07/19/2013 16:58:9 Page 15

ACKNOWLEDGEMENTS

Canada Research Chairs, Natural Science and Engineering Research Council,

Saskatchewan Agriculture Development Fund, Saskatchewan Pulse Growers,

and Brewing and Malting Barley Research Institute, Winnipeg are gratefully

acknowledged for supporting seed carbohydrate research in our laboratory.

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2 Type 2 Resistant Starch inHigh-Amylose Maize Starchand its Development

Hongxin Jiang and Jay-lin JaneDepartment of Food Science and Human Nutrition, Iowa State University, USA

2.1 INTRODUCTION

Starch is a reserve carbohydrate widely present in seeds, roots, tubers, leaves,

stems and fruits of plants. Most starch consists of two polymers – amylopectin

and amylose – which are organized in a semi-crystalline granular structure

(Jane, 2004). Amylopectin has highly branched structures, consisting of linear

chains of a(1! 4)-linked a-D-glucopyranose units, which are connected by

approximately 5% a(1! 6) glycosidic branch linkages (French, 1984).

Amylose is an essentially linear polymer of a(1! 4)-linked a-D-glucopyr-anose units (Takeda et al., 1986, 1989, 1993).

A third component, known as intermediate component (IC), is present in

maize mutants such as high-amylose and sugary-1 maize starches. IC has

branched structures with molecular weights smaller than amylopectin but

similar to amylose (Baba & Arai, 1984; Klucinec & Thompson, 1998; Li

et al., 2008; Wang et al., 1993). The IC molecules also possess iodine-binding

capacities and b-amylolysis limits between amylopectin and amylose mol-

ecules (Kasemsuwan et al., 1995). Normal maize starch contains about 30%

amylose (Hasjim et al., 2009). High-amylose maize starches with amylose

contents of 50%, 70% and 80% have been commercialized. High-amylose

maize starch with 90% amylose has also been reported (Shi et al., 1998).

Starch is present invarieties of foods and is amajor energy source for humans.

After ingesting, starch is partially hydrolyzed by human salivary a-amylase and

is predominantly digested in the small intestine by pancreatic a-amylase and

mucosal enzymes (Sang & Seib, 2006). Starch from different botanical sources

has different digestive rate and total digestibility, which results in different

physiological effects on human health. Resistant starch (RS) is a portion of starch

23




CH02GR 07/30/2013 17:4:49 Page 24

in foodswhichcannot behydrolyzedbyenzymes in the small intestine andpasses

on to the large intestine as a prebiotic (Englyst & Cummings, 1985; Englyst &

Macfarlane, 1986). RS provides many health benefits to humans, as previously

reported (Behall & Howe, 1996; Behall et al., 2006a, 2006b; Dronamraju et al.,

2009;Higgins et al., 2004; Pawlak et al., 2004; Robertson et al., 2003, 2005;Van

Munster et al., 1994; Zhang et al., 2007).

Enzymatic hydrolysis of raw starch granules is a complex process. Many

characteristics of the starch determine the rate and the extent of enzymatic

hydrolysis of the granules, such as granular size, structure of the amylopectin,

polymorph, amylose content and lipid content. These characteristics are species

dependent andare also affectedbygrowth conditions andpost-harvest treatment

(Jane, 2006;Lehmann&Robin, 2007; Setiawan et al., 2010;Tester et al., 2006).

It is well known that starch granules with larger granular sizes are digested at a

slower rate than those with smaller granular sizes, because larger starch granules

have relatively smaller surface area for enzymes to attack (Tester et al., 2006).The

A-type polymorphic starch granules, such as normal andwaxymaize, rice,wheat,

and taro starch, are known to have ‘weak points’ and are more easily hydrolyzed

by enzymes than are the B- and C-type polymorphic starch granules such as

potato, high-amylosemaize, green banana and pea starch (Jane et al., 2003; Jane,

2007). This difference is attributed to the branch chain-length of amylopectin and

the packing of double helices in the starch granule (Figure 2.1).

B type A type

2.1 nm

6.9 nm

1.6 nm

7.4 nm 9.0 nm

C

A

C

A

C

A

Figure 2.1 Structure models of amylopectins for A type and B type starches.A: amorphous; C: crystalline. Reproduced from Carbohydrate Polymers, 49(3), YooS.-H. and Jane J.-l. Molecular weights and gyration radii of amylopectins determinedby high-performance size-exclusion chromatography equippedwithmulti-angle laser-light scattering and refractive index detectors, 307–314. Copyright 2002, withpermission from Elsevier.

24 Resistant Starch

CH02GR 07/30/2013 17:4:49 Page 25

The amylopectin of the A-type polymorphic starch has a larger

proportion of short A and B1 chains but smaller numbers of long B2,

B3, and B4 chains than that of the B-type polymorphic starch (Jane et al.,

1999). B2, B3, and B4 chains extend through multiple clusters of

amylopectin and their movements are constrained by the consecutive

crystalline regions in the starch granule. The short A and B1 chains,

however, extend through one cluster of amylopectin and have free ends to

move around within the cluster and to form closely packed monoclinic

crystallites. Consequently, the A-type polymorphic starch granules consist

of voids (weak points) (Figure 2.2a,c; Jane, 2006; Jane, 2007; Jane et al.,

2004; Pan & Jane, 2000), which are easily penetrated and hydrolyzed by

Figure 2.2 Internal structures of starch granules. a (maize) and b (potato): scanningelectron micrographs of inner part of granules remaining after removal of thechemical gelatinized granule surface. Reproduced from Jane, 2006; 2007. c and d:confocal laser-scanning micrographs of normal maize and potato starch granules,respectively; starch granules were stained using Rhodamine B dye. Reproduced fromJane, 2006.

Type 2 Resistant Starch in High-Amylose Maize Starch and its Development 25

CH02GR 07/30/2013 17:4:52 Page 26

enzymes to generate pin holes and channels (Fannon et al., 1992, 1993;

Jane, 2006).

The B-type polymorphic starch granules, consisting of rigidly organized

amylopectin branch chains (Figure 2.1) and without weak points or voids

(Figure 2.2b,d) (Jane, 2006; Jane, 2007; Jane et al., 2004; Jane & Shen, 1993),

are normally digested slowly by erosion of starch granules from the surface

(Jane, 2006; Robyt, 1998). After cooking, potato starch, however, is highly

digestible. The increase in amylose content in normal starch granules (Jane

et al., 2003) and in high-amylose starch granules (Evans & Thompson, 2004;

Jiang et al., 2010b; Li et al., 2008; Shi & Jeffcoat, 2001) reduce the rate of

enzymatic hydrolysis of the raw starch granules. These features can be

attributed to the fact that, in the normal starch granule, the amylose is more

concentrated at the periphery of starch granules and interacts with amylopectin

to form a hard shell (Debet & Gidley, 2007; Jane & Shen, 1993; Pan & Jane,

2000); this makes the starch granule more resistant to enzymatic hydrolysis

(Tester et al., 2006). In high-amylose maize starch, amylose forms crystalline

structure, which is highly resistant to enzymatic hydrolysis (Evans & Thomp-

son, 2004; Jiang et al., 2010b; Li et al., 2008; Shi & Jeffcoat, 2001).

In general, starch is consumed by humans after cooking. Starch gelatini-

zation during cooking is an irreversible reaction which is associated with the

dissociation of double helices and loss of birefringence viewed under a

polarized light (Jane, 2004). Gelatinization properties of starch isolated

from different botanical origins differ, and this relates to starch molecular

structures and minor components present in the starch granule (Jane, 2004;

Yoo et al., 2009). The enzymatic hydrolysis of gelatinized starch (amorphous)

is much faster than that of raw starch granules (semi-crystalline) (Lehmann &

Robin, 2007; Tester et al., 2006).

High-amylose maize (amylose-extender, aemutant) starches with amylose

contents ranging from 50–80% have been commercially available and are

widely used in food and non-food applications (Fergason et al., 1994;

Richardson et al., 2000). Maize ae single-mutant starch contains about

65% apparent amylose and about 15% RS (Li et al., 2008) – substantially

higher than normal (non-mutant) maize starch (�30% and �1.5%, respec-

tively) (Hasjim & Jane, 2009; Hasjim et al., 2009). After cross-breeding the

maize ae-mutant with a maize line carrying high-amylose modifier (HAM)

gene, the apparent amylose content of the starch (GEMS-0067) elevates to

�85% and the RS content of the starch increases to �43% (Li et al., 2008).

Maize ae-mutant starch consists of spherical and elongated starch

granules (Figure 2.3a,b), which differ from the normal maize starch in

consisting of spherical and angular granules (Figure 2.3e; Hasjim et al.,

2009; Jiang et al., 2010b; Li et al., 2007; Mercier et al., 1970; Perera et al.,

26 Resistant Starch

CH02GR 07/30/2013 17:4:52 Page 27

2001; Wolf et al., 1964). The elongated starch granule content of a double

mutant of ae and HAM genes, GEMS-0067 line, increases up to 32%

(Figure 2.3a; Jiang et al., 2010b). These elongated starch granules retain

their granular shapes after cooking at 95–100 �C with excess water and are

highly resistant to enzymatic hydrolysis (Figure 2.3c). In this chapter,

formations of RS and elongated starch granules in the maize ae-mutant

starch and how the effect of HAM gene on the RS content of the maize

ae-mutant starch will be discussed.

Figure 2.3 Scanning electron micrographs of maize starches and resistant-starch(RS) residues (Jiang et al., 2010b). (a) GEMS-0067 starch (�85% apparent amylose)showing spherical (s) and elongated (e) granules. (b) H99ae starch (�65% apparentamylose). (c) GEMS-0067 RS residue remaining after enzymatic hydrolysis at 95–100 �C (AOAC Method 991.43). (d) H99ae RS residue in a gel-like form. Arrowsindicate fragmented, hollowed and half-shell-like granules. (e) Normal maize starch.Reprinted from Jiang et al. (2010b), Copyright 2010, with permission from Elsevier.


CH02GR 07/30/2013 17:4:52 Page 28

2.2 RS FORMATION IN HIGH-AMYLOSE MAIZESTARCH

Although high-amylose maize starch has been developed for over 60 years

(BeMiller, 2009; Kramer et al., 1956; Vineyard & Bear, 1952; Vineyard et al.,

1958), interests in the RS of the high-amylose maize starch has just emerged in

the past two decades, resulting from consumer awareness of health food

benefits (Sajilata et al., 2006). The structures and properties of the high-

amylose maize starch and its RS residue have been intensively studied in order

to understand the RS formation in the starch (Evans & Thompson, 2004; Jiang

& Liu, 2002; Jiang et al., 2010b; Li et al., 2008; Shi et al., 1998; Shi &

Jeffcoat, 2001). It has been reported that molecular weight and branch chain

length distributions of RS residues remaining after pancreatic a-amylase

hydrolysis are similar to that of native starch counterparts (Evans &

Thompson, 2004; Shi & Jeffcoat, 2001).

After enzymatic hydrolysis of high-amylose maize starches at 95–100 �C(AOAC Method 991.43), the RS residues display semi-crystalline structures

with the B-type polymorph (Jiang et al., 2010b). The onset gelatinization

temperatures of the RS residues are above 100 �C, indicating the long-chain

double-helical crystallites are present in the RS residues. Gel-permeation

chromatograms show that the RS residues consist of large molecules with

degrees of polymerization (DP) 840–951 and small molecules with DP 59–

74 (Jiang et al., 2010b). This differs from molecular weight distributions of

RS residues remaining after pancreatic a-amylase hydrolysis of the native

high-amylose maize starches (Evans & Thompson, 2004; Shi & Jeffcoat,

2001).

The molecular weight distributions of the debranched RS residues remain-

ing after enzymatic hydrolysis of the high-amylose maize starches at

95–100 �C show that the RS residues consist mostly of linear molecules

and are mainly derived from amylose/IC molecules (Jiang et al., 2010b).

Thus, it is concluded that the RS residues of high-amylose maize starches

consist of mainly semi-crystalline structure of long-chain double-helices of

amylose/IC (Jiang et al., 2010b). It is likely that the amylose/IC crystallites in

native high-amylose maize starch are present in blocks, which prevent the

starch granule from swelling at 95–100 �C, maintain the semi-crystalline

structure and protect the bulk of the amylose and IC and a small portion of

amylopectin molecules from enzymatic hydrolysis (Jiang et al., 2010b).

The presence of amylose double-helical crystallites in high-amylose maize

starch is also supported by the structure and properties of the Naegeli dextrin,

which is produced by hydrolyzing the starch at 38 �C using 15.3% sulphuric

28 Resistant Starch

CH02GR 07/30/2013 17:4:52 Page 29

acid (Jiang et al., 2010e). The Naegeli dextrins of high-amylose maize

starches consist of double helices with chain lengths of DP� 12

(8.6–11.2%), DP 13–24 (38.7–54.1%), DP 25–36 (23.6–30.6%) and DP� 37

(13.1–22.1%). Because the longest detectable chains of the Naegeli dextrin

of ae waxy maize starch are DP 25 (Jane et al., 1997), the long-chain double

helices of DP� 25 present in the Naegeli dextrin are attributed to crystalline

fragments of amylose double helices (Jane & Robyt, 1984; Jiang et al.,

2010b, 2010e). The thermal properties of the Naegeli dextrins of the high-

amylose maize starches display very high peak (113.9–122.2 �C) and

conclusion gelatinization-temperatures (148.0–160.0 �C), which also sup-

port the presence of amylose double helices (Jiang et al., 2010e; Sievert &

Pomeranz, 1989, 1990).

Although high-amylose maize starches contain approximately 0.2–0.7%

lipids (Jiang et al., 2010b; Morrison, 1992, 1993, 1995; Morrison et al.,

1993), the lipids present in the starch granules also reduce the enzyme

digestibility of the starch at 95–100 �C (Jiang et al., 2010b). After the lipids

are removed from the starch granules, the RS contents of the high-amylose

maize starches reduce from 10.6–43.4% to 9.0–28.9%. The effects of lipids

on the RS content of the high-amylose maize starch are discussed in other

chapters of this book.

2.3 RS FORMATION DURING KERNEL DEVELOPMENT

Although RS and amylose/IC double-helical crystalline structures are found

in mature high-amylose maize starch granules after harvesting and drying

(Jiang et al., 2010b), it is not known whether the RS and the amylose/IC

double-helical crystalline structure are formed during kernel development

or are induced during post-harvest processing and boiling/a-amylase

hydrolysis.

Jiang et al. (2010a) studied RS formation during kernel development of

GEMS-0067 line, a maize double-mutant of ae and HAM genes. Gelatiniza-

tion thermograms of the starches harvested at 15, 20, 30, 40, 54 (mature) days

after pollination (DAP) display a major thermal transition (first peak) with the

peak temperature between 76.6–81.0 �C and an additional thermal transition

(second peak) with the peak temperature �97.1 �C (Figure 2.4). The second

peak first appears as a shoulder on 20 DAP and gradually increases into a

significant peak on 30, 40, and 54 DAP (Jiang et al., 2010a). The relative

intensity of the first peak (76.6–81.0 �C) decreases with kernel maturation and

a decrease in the amylopectin content of the starch, indicating that this peak

corresponds to the dissociation of amylopectin crystallites (Jiang et al., 2010a,


CH02GR 07/30/2013 17:4:53 Page 30

2010b; Kasemsuwan et al., 1995; Li et al., 2008). The increase in the size of

the second peak correlates with the lipid content of the starch, and this peak

disappears after removal of lipids from the starch, indicating that this

additional peak is the melting of amylose-lipid complex (Jiang et al.,

2010a; Tufvesson et al., 2003a, 2003b).

After removal of lipids, the conclusion gelatinization-temperature of the

starch does not change. The conclusion gelatinization temperature, however,

increases with kernel maturation, from 105.0 �C on 15 DAP to 117.8–122.2 �Con later dates (Jiang et al., 2010a). The percentage of the enthalpy change of

the thermal transition at temperature range above 95 �C, which corresponds tothe melting of long-chain double-helical crystallites of amylose/IC, increases

with the kernel maturation and significantly correlates with the RS content of

the starch (Jiang et al., 2010a). The RS content of the high-amylose maize

starch increases with kernel maturation and directly correlates with the

amylose/IC content of the starch (r¼ 0.99, p< 0.001) (see Table 2.1).

Thus, the authors conclude that the long-chain double-helical crystallites

of amylose/IC in the high-amylose maize starch develop with the kernel

maturation, which results in an increase in the RS content of the starch (Jiang

et al., 2010a).

140130120110100908070605040

Hea

t Flo

w E

ndo

Up

Temperature (°C)

15 DAP

20 DAP

30 DAP

40 DAP

54 DAP

Figure 2.4 Differential scanning calorimetry (DSC) thermograms of native GEMS-0067 starches harvested at different developmental stages (Jiang et al., 2010a).DAP: days after pollination. The peak area above the dashed line indicates meltingof amylose-lipid complex. Reprinted with permission from Jiang et al. (2010a).Copyright 2010 American Chemical Society.

30 Resistant Starch

CH02GR 07/30/2013 17:4:53 Page 31

2.4 ELONGATED STARCH GRANULES OFHIGH-AMYLOSE MAIZE STARCH

2.4.1 Structures of elongated starch granules

Different from normal maize starch, which contains starch granules with

spherical and angular shapes (Figure 2.3e; Hasjim et al., 2009; Perera et al.,

2001; Wongsagonsup et al., 2008), high-amylose maize starch contains

elongated granules in addition to spherical granules (Figure 2.3a,b; Boyer

et al., 1976; Jiang et al., 2010b; Mercier et al., 1970; Shi & Jeffcoat, 2001;

Sidebottom et al., 1998; Wolf et al., 1964). Starch of maize double mutant

GEMS-0067 consists of up to 32% elongated granules, which is substantially

greater than that of the maize ae single-mutant starch (�7%) and normal

maize starch (0%) (Campbell et al., 2007; Hasjim et al., 2009; Jiang et al.,

2010b; Li et al., 2007; Perera et al., 2001; Wongsagonsup et al., 2008). The

percentage of elongated starch granules increases with kernel maturation and

the increase in amylose/IC content of the starch (Jiang et al., 2010a, 2010b,

2010d; Mercier et al., 1970). Elongated starch granules are mostly found in

mature endosperm cells located in the central part of the crown region of the

high-amylose maize kernel (Boyer et al., 1976).

Starch molecules in the granule are present in semi-crystalline structures of

double helices. The birefringence pattern of a starch granule observed under a

polarized light microscope reflects the arrangement of starch molecules in the

granule (French, 1984). Normal maize starch granules display a typical

Table 2.1 Resistant starch (RS) and amylose/intermediate component (IC) contents ofGEMS-0067 starches harvested at different kernel-developmental stages (Jiang et al.,2010a). Reprinted with permission from Jiang et al. (2010a). Copyright 2010 AmericanChemical Society.

Sample RSa (%) Amylose/ICb (%)

15 DAPc 9.0�1.2 55.2�0.520 DAP 26.4�0.1 78.4�0.230 DAP 29.6�0.8 81.9�0.340 DAP 32.0�0.1 88.6�1.354 DAP 32.1�0.3 87.6�0.4Coefficient between RS and amylose/IC content 0.99d

aRS content of starch was determined using AOAC method 991.43 for total dietary fibre.bAmylose/IC content of starch was determined using Sepharose CL-2B gel-permeation chromatog-

raphy followed by the total carbohydrate (phenol-sulphuric acid) determination.cDAP: days after pollination.dp<0.001.


CH02GR 07/30/2013 17:4:53 Page 32

Maltese cross birefringence pattern (Wongsagonsup et al., 2008), reflecting

that starch molecules in the granule are aligned radially from the hilum, which

are oriented perpendicular to the granule surface.

Most spherical granules of high-amylose maize starch exhibit a normal

Maltese cross birefringence (Figure 2.5, granule a), similar to the granules of

normal maize starch. This birefringence pattern is consistent with the confocal

laser-scanning micrograph (CLSM) of the 8-aminopyrene-1,3,6-trisulfonic

acid (APTS)-derivatized spherical granule, which shows a hilum with a bright

colour at the centre of the granule (Figure 2.6a; Glaring et al., 2006; Jiang

et al., 2010c). These findings indicate that the spherical granule is developed

from one single granule nucleus.Elongated starch granules, however, display three different types of

birefringence patterns (Figure 2.5). The type 1 birefringence pattern displays

several Maltese crosses overlapping in one granule (Figure 2.5, granule b), while

the type 2 birefringence pattern shows one or moreMaltese crosses in one part of

the granule and weak or no birefringence on the rest of the granule (Figure 2.5,

granule c). The type 3 birefringence pattern exhibits a granule displayingweak or

no birefringence (Figure 2.5, granule d; Jiang et al., 2010c; Wolf et al., 1964).

The differences in birefringence patterns of elongated starch granules

indicate that arrangements of starch molecules in the granule differ between

elongated granules, and even between different parts of one elongated granule.

This feature is consistent with the CLSM images of APTS-stained elongated

granules, which showmultiple regions of different fluorescence intensity in one

elongated granule (Figure 2.6b–g) (Glaring et al., 2006; Jiang et al., 2010c).

Figure 2.5 Polarized (a) and phase contrast (b) light micrographs of high-amylosemaize (GEMS-0067) starch. Arrows indicate birefringence patterns of: (a) onegranuledisplaying one Maltese cross; (b) Maltese crosses overlapping in one granule; (c) asinglegranuleconsistingofoneormoreMaltesecrossesandweak/nobirefringenceonthe rest part of the granule; (d) granule showingweak/no birefringence. Bar¼20mm.

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2.4.2 Formation of elongated starch granules

Starch granule formation in the amyloplast of high-amylose maize endosperm

at an early stage of kernel development (20 DAP) has been studied using

transmission electron microscopy (TEM) (Jiang et al., 2010c). Some amy-

loplasts at the subaleurone layer of high-amylose maize endosperm contain

only one spherical granule showing normal growth rings and a hilum

(Figure 2.7a), which are similar to amyloplasts in normal maize endosperm.

The majority of amyloplasts, however, contain two or more small starch

granules (Figure 2.7b).

These small granules in the amyloplast, which develop at the early stage,

fuse and grow into an elongated starch granule (Figures 2.7c–g; Jiang et al.,

2010c). This fusion is likely the result of interactions between the amylose of

adjacent granules, because high-amylose maize starch contains a large

concentration of amylose. The amylose molecules of one granule can contact

that of an adjacent granule and form anti-parallel double helices between the

Figure 2.6 Confocal laser-scanning microscope images of high-amylose maize(GEMS-0067) starch granules (Jiang et al., 2010c). Starch granules were labelled using8-aminopyrene-1,3,6-trisulfonic acid APTS). (a) Spherical granule showing brightcolour around the hilum; (b), (c), (d), (e), (f), and (g) show elongated starchgranules displaying multiple regions with intense fluorescence. Bar¼10mm. Reprintedfrom Jiang et al. (2010c), Copyright 2010, with permission from Elsevier.


CH02GR 07/30/2013 17:4:56 Page 34

two granules (Figure 2.7d; Jiang et al., 2010a, 2010b; Li et al., 2008), which

bind the two adjacent granules and prevent the amyloplast from dividing.

Eventually, the two granules grow into one elongated granule (Figure 2.7e).

A proposed mechanism of the elongated starch granule formation is shown

in Figure 2.8. The mechanism is consistent with the fact that the number of

elongated starch granules increases with the increase in the amylose content of

the starch (Jiang et al., 2010a, 2010b). In the normal maize starch, amylose

content is low (�30%) and branch chains of amylopectin are short comparing

with amylose. The short branch chains of amylopectin cannot form stable anti-

parallel double helices with branch chains of an adjacent granule in a normal

maize amyloplast during granule development. Thus, the small starch gran-

ules in the normal maize amyloplast are separated with the division of

Figure 2.7 Transmission electron micrographs of high-amylose maize (GEMS-0067) endosperm tissue harvested on 20 days after pollination (Jiang et al.,2010c).(a) Spherical starch granule with a hilumat centre of granule and growth rings.(b)Overviewofendosperm tissue. (c) Twostarchgranules initiated inoneamyloplastatearly stage of starch granule development. (d) Initial fusion of starch granules. (e) Twofused starch granules forming an elongated starch granule. (f) Two connected starchgranules and a third starch granule in the amyloplast protrusion. (g) Three smallgranules fused into one granule, with one small granule at head showing hilum andgrowth rings and the other two granules displaying no hilum or growth rings.m:membrane boundary of amyloplast. Black arrow indicates hilum. Bars¼1mm onA, C, D, F and 2mm on B, E, G. Reprinted from Jiang et al. (2010c), Copyright2010, with permission from Elsevier.

34 Resistant Starch

CH02GR 07/30/2013 17:5:0 Page 35

amyloplast (Jiang et al., 2010c). Most amyloplasts in normal maize endo-

sperm consist of a single starch granule.

2.4.3 Location of RS in the starch granule

The RS residues of GEMS-0067, consisting of about 85% amylose, are found

mostly in elongated granular structures (Figure 2.3c), whereas that of the ae

single-mutant, consisting of approximately 65% amylose, are present in a gel

form (Figure 2.3d; Jiang et al., 2010b). This is attributed to a greater content of

amylose/IC double-helical crystallites present in the granules of GEMS-0067,

particularly elongated granules (Jiang et al., 2010b).

The hollowed and half-shell-shaped granules and the gel-like starch

(Figure 2.3c,d) observed in the RS residues are the remnants of the outer

layer of spherical granules (Jiang et al., 2010b). These structures can be

attributed to the fact that the amylopectin molecules around the hilum are

loosely packed and can be promptly gelatinized and hydrolyzed by thermally

stable a-amylase at 95–100 �C. Amylose molecules, however, are more

concentrated at the periphery of the spherical granules (Jane & Shen,

1993; Jiang et al., 2010a; Jiang et al., 2010b; Li et al., 2007; Pan & Jane,

2000). The amylose molecules at the periphery of the starch granules interact

between themselves and with amylopectin molecules to form a hard shell

Figure 2.8 Proposed formation mechanism of elongated starch granule in theamyloplast (Jiang et al., 2010c). (a) Two starchgranules, eachwith hilumandgrowthrings, are initiated in amyloplast at an early stage of granule development. (b) Twogranules begin fusion through amylose interaction by forming anti-parallel doublehelices, which prevent amyloplast division. (c) Fused granules have integrated outerlayer growth rings.m:membrane boundary of amyloplast. Reprinted from Jiang et al.(2010c), Copyright 2010, with permission from Elsevier.


CH02GR 07/30/2013 17:5:1 Page 36

which is difficult to disperse and, thus, is resistant to enzymatic hydrolysis

(Debet & Gidley, 2007; Gray & BeMiller, 2004; Huber & BeMiller, 2001;

Jane & Shen, 1993; Jiang et al., 2010a, 2010b; Li et al., 2007; Pan & Jane,

2000).

2.5 ROLES OF HIGH-AMYLOSE MODIFIER (HAM)GENE INMAIZE ae-MUTANT

The role of HAM gene and its dosage effects on the physicochemical

properties of maize ae-mutant starch have been studied and reported (Jiang

et al., 2010d). Maize endosperm is a triploid tissue, resulting from the

fertilization of two maternal nuclei and one paternal nucleus (Birchler,

1993). Thus, endosperms with 0, 1, 2, and 3 doses of HAM gene are produced

by self- and inter-pollination of an ae single-mutant line and the ae and HAM

double-mutant line, GEMS-0067. The increase in HAM gene dosage in the

maize ae mutant increases the apparent amylose content of the starch (see

Table 2.2; Jiang et al., 2010d). After including three doses of HAM gene to the

maize ae-mutant, the starch has longer branch chain-lengths of IC molecules

but slightly shorter branch chain-lengths of amylopectin molecules than

maize ae single-mutant starch (Li et al., 2008). Two or three doses of

HAM gene in maize ae-mutant substantially increase the RS content of

the maize ae-mutant starch. One dose of HAM gene, however, has little effect

on the RS content.

Morphological properties of starch granules are also influenced by the

dosage of HAM gene (Jiang et al., 2010d). The greater the HAM gene dosage

in the starch, the greater the number of elongated granules is found in

maize ae-mutant starch. With the presence of three doses of HAM gene,

many maize ae-mutant starch granules display weak or no birefringence,

Table 2.2 Amylose and resistant starch (RS) contents of native maize starches isolatedfrom kernels of self- and intercrossed lines GEMS-0067 (G) and H99ae (H) (Jiang et al.,2010d). Reprinted with permission from Jiang et al. (2010a). Copyright 2010 AmericanChemical Society.

Sample HAM gene doses Amylosea (%) RSb (%)

G/G 3 69.9�0.0 35.0�0.5G/H 2 64.9�0.4 28.1�0.8H/G 1 61.1�0.1 12.9�0.2H/H 0 56.3�0.4 15.7�0.2

aAmylose content of starch was determined using iodine-colorimetric method.bRS content of starch was determined using AOAC method 991.43 for total dietary fibre.

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CH02GR 07/30/2013 17:5:1 Page 37

indicating changes in molecular organization of the granules. The increase in

HAM gene dosage decreases the percentage crystallinity of starch, which is

consistent with the decrease in amylopectin content of the starch (Jiang et al.,

2010d). This is attributed to amylopectin having short branch chains which are

free to move and interact with one another and, thus, facilitate the formation of

double-helical crystallites.

Maize ae-mutant starches display multiple thermal-transitions during

gelatinization (Jiang et al., 2010a, 2010b, 2010d). The temperature of the

thermal transition range increases with the increase in the HAM gene dosage.

The first thermal transition peak, with the peak temperature at �82.9 �C,corresponds to the melting of amylopectin crystallites (Jiang et al., 2008;

Kasemsuwan et al., 1995; Li et al., 2008), which decreases with an increase in

HAM gene dosage (Jiang et al., 2010d). The second thermal transition peak,

with the peak temperature at �99.1 �C, corresponds to the dissociation of

amylose-lipid complexes (Jiang et al., 2010a; Li et al., 2008; Tufvesson et al.,

2003a, 2003b), which increases with an increase in HAM gene dosage (Jiang

et al., 2010d). The conclusion gelatinization-temperature of the starch is

above 100 �C and increases with the increase in HAM gene-dosage (Jiang

et al., 2010d), indicating the formation of amylose/IC crystallites (Jiang et al.,

2010a).

These findings indicate that the formation of amylose/IC crystallites and

amylose-lipid complex increasewith the increase in HAMgene dosage, which

is consistent with the increase in the RS content of the starch (Jiang et al.,

2010d).

2.6 CONCLUSIONS

Long-chain double-helical crystallites of amylose/IC in high-amylose maize

starch are developed during kernel development and are concentrated in the

elongated granules and at the outer layer of the spherical granules. The

amylose/IC crystallites, having onset gelatinization temperature above

100 �C, restrict starch granules from swelling and dispersion at 95–

100 �C and protect the starch molecules from enzymatic hydrolysis at

95–100 �C. Lipids present in the granule also protect starch granules

from enzymatic hydrolysis at 95–100 �C. The formation of elongated starch

granules in high-amylose maize starch results from amylose interactions

between adjacent small granules in the amyloplast during granule develop-

ment. The inclusion of HAM gene(s) to the maize ae-mutant increases the

crystallites of amylose/IC in the starch, which results in the increase in RS

content of the starch.


CH02GR 07/30/2013 17:5:1 Page 38

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3 RS4-Type Resistant Starch: Chemistry,Functionality and Health Benefits

Clodualdo C. Maningat1 and Paul A. Seib2

1MGP Ingredients, Inc., USA; Department of Grain Science and Industry,Kansas State University, USA

2Department of Grain Science and Industry, Kansas State University, USA

3.1 INTRODUCTION

The statistics on obesity are alarming; more than one-third of adult Americans

and almost 17% of youth were classed as obese in 2009–2010 (Ogden et al.,

2012). One television news report disturbingly labelled overweight youth as

‘coronary time bombs’, as they are likely to develop heart disease when they

grow into adulthood. As a potential consequence, billions of dollars will be

spent in weight-related bills. The high price tag is simply because obesity is

not a singular medical condition. Being overweight or obese is also associated

with Type 2 diabetes, cardiovascular disease, high cholesterol, hyperlipidae-

mia, hypertension, stroke, cancer and other diseases. About one-third of

Americans have hypertension – a major risk factor for heart disease and stroke

if left untreated.

Resistant starch (RS), as a component of dietary fibre, is not only beneficial

but can be considered essential to the general health and well-being of

consumers worldwide. It demonstrates numerous positive physiological

effects and, therefore, plays a critical role as a food ingredient in addressing

the widespread metabolic syndrome that afflicts the general population. This

chapter specifically highlights the properties of RS belonging to the RS4

classification and underscores its effectiveness in alleviating diet and lifestyle-

related diseases.

43




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3.2 HISTORICAL ACCOUNT OF STARCHINDIGESTIBILITY

From the early 1900s into the 1930s, scientists explored the in vitro and in vivo

amylase digestibility of raw and gelatinized starches from different botanical

sources (Fofanow, 1911; Thorpe, 1913; Daniels & Strickler, 1917; Langwor-

thy & Deuel, 1920, 1922; Katz, 1934) (Table 3.1). Those researchers

succeeded in developing two of the concepts that we know today about

RS: that several raw starches, especially raw potato starch, are resistant to

digestion; and that retrograded starch, which was then termed as

‘amylocoagulose’ (Katz, 1934), resisted digestion.

The linkage between dietary fibre and starch as a dietary source of fibrewas

established in 1982, when British researchers discovered the presence of

starch ‘resistant’ to digestion in the residue of cooked and cooled potatoes

recovered after amylase hydrolysis during the determination of non-starch

polysaccharides (Englyst et al., 1982). A decade after Englyst’s work, a

formal definition of resistant starch as ‘the sum of starch and products of

starch degradation not absorbed in the small intestine of healthy individuals’

was established (Asp, 1992). Then, in 2001, a revised definition of dietary

fibre, which included RS under analogous carbohydrates, was introduced by

the American Association of Cereal Chemists (2001).

Subsequently, other organizations and countries followed suit (Institute of

Medicine, 2001; European Food Safety Authority, 2007) and, finally in 2009,

the Codex Alimentarius Commission adopted a new dietary fibre definition

that included RS under carbohydrate polymers obtained from food raw

material by physical, enzymatic or chemical means (Codex Alimentarius

Commission, 2009). In 2012, Health Canada, after several years of delibera-

tion, issued a new dietary fibre definition that is consistent with the definition

adopted by Codex Alimentarius Commission, and includes RS under novel

fibres (Health Canada, 2012).

RS4-type resistant starch belongs to one of the five types or classes of RS.

Englyst & Macfarlane (1986), citing the work of Englyst (1985), reported on

the classification of starch for nutritional purposes into readily digestible

starch and resistant starch, which was further classified into RS1, RS2, RS3a,

and RS3b. The preceding two types of RS3 were differentiated by their

composition as being mainly staled amylopectin and mainly retrograded

amylose, respectively.

Englyst & Cummings (1987) proposed a classification of starch based on

its digestibility that can distinguish readily digestible starch in freshly cooked

foods, partially resistant starch in raw potato and banana starch, and resistant

44 Resistant Starch

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Table 3.1 Chronology of events leading to resistant starch, its classification to RS1-RS5and its inclusion in the definition of dietary fibre.

Year Events References

1911 Raw potato starch was less digested inhuman subjects, but raw wheat, oat andrice starches were practically assimilated.

Fofanow, 1911

1917 Taka-diastase indigestible starch was foundin bakery products.

Daniels and Strickler,1917

1920–1922 Raw corn, wheat, cassava, rice and taro rootstarches were completely digested in vivo,but raw potato, arrowroot and cannastarches were less digestible.

Langworthy and Deuel,1920, 1922

1934 ‘Amylocoagulose’, a retrograded material inannealed starch gels, was not digested bymalt extract.

Katz, 1934

1953 The word ‘dietary fibre’ was first used todescribe plant cell walls in the diet.

Hipsley, 1953

1961–1962 Raw high-amylose corn starch was resistantto in vitro and in vivo digestion.

Ackerson, 1961; Leach& Schoch, 1961;Borchers, 1962;Sandstedt et al., 1962

1969–1971 Chemically modified starch was resistant todigestion by pancreatin.

Janzen, 1969; Leegwater& Luten, 1971

1971–1973 Prevalence of ‘Western’ diseases indeveloped societies, but not in Africa, wasattributed to inadequate dietary fibre(non-starch polysaccharides) intake.

Burkitt, 1971, 1973a,b;Trowell, 1972

1977 Raw banana starch resisted in vitro digestionby a-amylase.

Fuwa, 1977

1981 Protocol for measuring glycemic index wasestablished.

Jenkins et al., 1981

1982 The term ‘resistant starch’ was coined forretrograded starch found in cooked andcooled potatoes.

Englyst et al., 1982

1984 Retrograded amylose was resistant todigestion by a-amylases and yieldedresistant fragments with degree ofpolymerization of 43–50.

Jane and Robyt, 1984

1986 Starch was classified for nutritional purposesinto readily digestible starch and resistantstarch, which was further classified intoRS1, RS2, RS3a and RS3b.

Englyst & Macfarlane,1986

1992 Official definition for ‘resistant starch’ wasestablished as ‘the sum of starch andproducts of starch degradation that arenot absorbed in the small intestines ofhealthy individuals’.

Asp, 1992

(continued)

RS4-Type Resistant Starch: Chemistry, Functionality and Health Benefits 45

3GCH03 07/19/2013 17:21:13 Page 46

Table 3.1 (Continued)

Year Events References

1992 The in vitro Englyst method to measurerapidly digestible starch, slowly digestiblestarch and resistant starch (by difference)was established.


1992 RS1, RS2 and RS3 classification of resistantstarch was introduced.


1993 Hi-MaizeTM, the first commercially availableresistant starch ingredient from high-amylose maize starch, was released.

Brown et al., 1995

1993 Chemically modified starch was alluded toas another form of resistant starch.

Eerlingen et al., 1993

1995 Chemically modified starch wasindependently reported by twolaboratories to represent RS4classification.

Eerlingen and Delcour,1995; Brownet al., 1995

1995–1996 The Englyst method for in vitro assay ofresistant starch was validated in vivo.

Silvester et al., 1995;Englyst et al., 1996

2000–2001 Low prevalence of ‘Western’ diseases amongAfricans was associated with adequateconsumption of resistant starch, ratherthan the non-starch polysaccharidecomponents of dietary fibre.

Ahmed et al., 2000;Topping & Clifton,2001

2001 American Association of Cereal Chemistsissued a revised definition of dietary fibreto include resistant starch underanalogous carbohydrates; Institute ofMedicine of the National Academy ofSciences issued a new definition for dietaryfibre, functional fibre and total fibre, withresistant starch falling under theclassification of functional fibre.

American Associationof Cereal Chemists,2001; Institute ofMedicine, 2001

2002 Direct method for quantifying resistant starchwas developed for RS1, RS2 and RS3.

McCleary et al., 2002

2006 RS5 classification was proposed to representamylose-lipid complexes organized in theV-type polymorph.

Brown et al., 2006

2009 Codex Alimentarius Commission issued anew definition for dietary fibre to includeresistant starch under carbohydratepolymers obtained from food raw materialby physical, enzymatic or chemical means.

Codex AlimentariusCommission, 2009

2010–2011 Methods to determine soluble, insoluble, andtotal dietary fibre (Codex definition) weredeveloped.

McCleary et al.,2010, 2011

2012 Health Canada issued a new definition fordietary fibre to include resistant starchunder novel fibres.

Health Canada, 2012

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starch formed as a result of food processing. RS was classified into three types

(RS1, RS2 and RS3) as discussed by Englyst et al. (1992):

� RS1 pertains to physically-entrapped starch present in whole or coarsely-

ground grains and pulses where the starch granules are encapsulated within

a cell wall so that amylase enzymes are prevented or delayed from having

access to the glycosidic bonds.� RS2 is raw starch with inherent enzyme resistance by virtue of its crystal-

line structure.� Recrystallized or retrograded amylose represents the RS3 type.

Eerlingen et al. (1993) alluded to the presence of another type of RS in

food, which they described as chemically modified starch to represent starch

that is rendered indigestible to amylase enzymes by virtue of their chemical

modification. Chemically modified starches can inhibit or reduce starch

digestibility by their functional groups blocking access of amylase enzymes,

or by the presence of atypical linkages such as (1! 2)- and (1! 3)-glycosidic

bonds that are not recognized by amylase enzymes.

A fourth type of RS (i.e. RS4) was introduced independently by Eerlingen

& Delcour (1995) and Brown et al. (1995) into the existing three types or

classes of RS. Brown et al. (2006) then added a fifth type of RS (i.e. RS5) to

represent amylose-inclusion complexes with lipids that exist as the V-type

crystals. It is worth noting that guest molecules such as emulsifiers and

surfactants can also form helical complexes with amylose and, to a lesser

extent, amylopectin (Krog, 1971; Evans, 1986; Eliasson, 1998; Faergemand&

Krog, 2006).

3.3 STARCHMODIFICATION YIELDING INCREASEDRESISTANCE TO ENZYME DIGESTIBILITY

Chemically modified food starches are traditionally used as ingredients in the

food industry to enhance the processing performance, physical attributes,

sensory properties and storage stability of consumer packaged food products.

Production of modified starches for food use includes acid treatment, oxida-

tion, esterification and etherification, or combinations of modification (Huber

& BeMiller, 2010; Mason, 2009).

Starch granules are penetrable by low molecular weight, water-soluble

solutes (or chemical reactants), but molecules larger than 1000 Daltons are

effectively excluded (Brown & French, 1977). The pathway of entry is

probably through the pores and channels located on the granule surface


3GCH03 07/19/2013 17:21:13 Page 48

(Huber & BeMiller, 2000; Kim & Huber, 2008; Han et al., 2006). These

granular features and other factors influence the chemical reactions occurring

in the granule.

Huber &BeMiller (2001, 2010) summarized the intrinsic factors (botanical

source, granule morphology, molecular structure, composition, starch prop-

erties) and extrinsic factors (reaction medium, reaction temperature, amount

of chemicals, type of chemicals, addition of salts, presence of catalysts) that

influence the chemical modification of starch. Biliaderis (1982) stated that

factors influencing the rate and selectivity of starch modification reactions at

the macromolecular level consisted of the reactivity of the starch hydroxyl

groups, specific area and organization of the starch granule, diffusion rate and

reactivity of the co-reactant, and steric factors due to bulkiness of the

substituent groups.

Other researchers discovered that cereal and legume starches are less

susceptible to structural modification than root and tuber starches (Singh

et al., 1993). Corn and amaranth starches displayed differences in optimum

reaction conditions during the preparation of sodium carboxymethyl deriv-

atives (Bhattacharya et al., 1995), while maize starches with a differing

amylose/amylopectin ratio exhibited variations in the distribution of hydrox-

ypropyl groups on the glucan chains (Azemi & Wooton, 1995).

Even though a combination of factors appears to dictate the rate and

selectivity of the modification reaction, Biliaderis (1982) and Hood&Mercier

(1978) suggested that the nature of a modifying agent is more important in

determining the substituent distribution in starch chains than the physico-

chemical characteristics of the granule. When the degree of substitution (DS)

increased to a relatively high value (e.g. DS¼ 0.85–2.89), a disorganization of

the granule crystal structure occurred (Chi et al., 2008). Small-angle X-ray

scattering measurements of acylated starches revealed that a longer substitu-

ent chain like butyrate is accommodated easily within the crystalline lamellar

structure, compared to acetate and propionate chains, leaving the nano-

structure practically unchanged (Lopez-Rubio et al., 2009).

At the molecular level, substituent groups are distributed unevenly (both

between starch molecules and over the length of molecules), indicating that

starch granules react heterogeneously (Huber & BeMiller, 2001, 2010). The

DS of the amylose fraction was much higher than that of the amylopectin

fraction, suggesting a difference in reactivity of amorphous and crystalline

regions (Huang et al., 2007).

Hoover & Sosulski (1986) hypothesized that derivatization sites for

phosphorus oxychloride (POCl3) occurred in the amorphous regions of the

granule, because no difference was observed between the x-ray diffractions of

the cross-linked and unmodified starches. Because POCl3 (or its first

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hydrolysis product, HOPOCl2) is highly reactive, it reacts with the first starch

molecules it encounters near the surface of the granules.

The location of modifying groups was proposed earlier by Biliaderis

(1982) when he introduced two possible structures for chemically modified

amylopectin. Fast-reacting reagents such as acetic anhydride or POCl3 tended

to react at or near the surface of a granule, where the tips (or non-reducing

ends) of the amylopectin cluster chains are located. Slow-reacting reagents

like propylene oxide tended to penetrate deeper to react not only near the tips

of amylopectin cluster chains, but also in the amylopectin region, where there

is dense branching. The location of phosphate cross-links may be assumed to

be uniformly distributed near the non-reducing ends and the branch points in

amylopectin, as noted by Manelius et al. (2000) for the reaction of a cationic

epoxide with potato starch. Both locations comprise the amorphous region of

the starch granule.

According to Huber & BeMiller (2000, 2001), POCl3 reaction with potato

and sorghum starches occurred at the granule surfaces, channels and cavities,

and likely within the granule matrix, as revealed by scanning electron

microscopy with compositional backscattered electron imaging. However,

POCl3 reacted with potato starch largely on the surface of the granule, because

potato starch granules lack pores and channels. It was observed that the

penetration of POCl3 through the perimeter surface of the granules inward was

somewhat impeded by a highly associated ‘outer shell’.

A number of researchers employing in vitro and in vivo techniques reported

that chemical modification of starches restricted to various degrees the

hydrolytic action of amylase enzymes. For example, modified raw or gelati-

nized starch with varying levels of cross-linking and/or substitution had

differential reduction of susceptibility to amylase action (Bjorck et al.,

1989; Hoover & Sosulski, 1986; Wolf et al., 1999; Wooton & Chaudhry,

1979; Conway & Hood, 1976; Janzen, 1969; Ackar et al., 2010; Klaushofer

et al., 1978; Xie & Liu, 2004; Xie et al., 2006; Wepner et al., 1999; Kim &

Huber, 2008; Leegwater & Luten, 1971; Azemi & Wooton, 1995; Hood &

Arneson, 1976; Hahn & Hood, 1980; Annison et al., 1995, 2003; Bajka et al.,

2007; Wolf et al., 2001; Wang et al., 2001; Woo & Seib, 2002).

On the basis of the above-mentioned studies, the mechanism of amylase

resistance of RS4 resistant starch can be summed up as follows. The

substituent groups along the a-1,4 D-glucan chains hinder enzymatic attack

and also make neighbouring glycosidic bonds resistant to degradation. The

presence of cross-linked polymer chains inhibits granular swelling and also

provides steric hindrance to the approach of the active site of amylase

enzymes. Moreover, cross-linking of starch may restrict the movement of

a-amylase through the granule surface pores and channels. Unusual


3GCH03 07/19/2013 17:21:13 Page 50

glycosidic bonds, such as a-1,2, a-1,3 or b-1,6 bonds, which form during

pyrolysis reaction on starch, are not substrates for amylases.

3.3.1 Cross-linked RS4 starches

Depending on the level of modifying chemicals, cross-linking of starch

granules from different botanical sources had the general effect of maintaining

granular integrity, restricting swelling, resisting mechanical shear and reduc-

ing paste viscosity (Huber & BeMiller, 2010; Mason, 2009). The degree of

cross-linking of starches used as thickeners in food products is too low to elicit

significant resistance to a-amylase hydrolysis (Wurzburg & Vogel, 1984;

Ostergard et al., 1988; Woo & Seib, 2002). Numerous cross-linking agents

have been reacted with starch, such as POCl3, sodium trimetaphosphate

(STMP), epichlorohydrin, adipic anhydride, citric acid and glutaric acid.

These inorganic and organic reagents are said to have bi- or poly-functional

reactive groups. That is, they react with two or more hydroxyl groups on the

starch molecular chains.

Early descriptions in the patent literature of cross-linking starch were those

of Felton & Schopmeyer (1943), utilizing 0.005–0.25% POCl3 at pH 8–12, and

Van Patten & Powell (1969), who improved the thickening ability of starch by

adding trisodium phosphate prior to the cross-linking reaction with POCl3.

POCl3-treated starch has a monoester : diester ratio of 1 : 3, based on the

thallium : phosphorus ratio assayed by inductively coupled plasma-atomic

emission spectroscopy (Koch et al., 1982). Chemical modifications such as

phosphate cross-linking inhibit alpha-amylase hydrolysis, as reported by

different researchers (Bjorck et al., 1989; Hoover & Sosulski, 1986; Wolf

et al., 1999;Wooton&Chaudhry, 1979; Conway&Hood, 1976; Janzen, 1969).

The presence of cross-linked residues across the glucan chains provides

steric hindrance to the action of amylase enzymes. Analysis of cross-linked

granular corn starch by a combination of gel permeation chromatography,

enzymatic hydrolysis and 31P-NMR confirmed that amylose was cross-linked

with amylopectin, no cross-linkages among amylose molecules existed, and

that amylose molecules were molecularly interspersed inside the granule

(Jane et al., 1992; Kasemsuwan & Jane, 1994).

Low levels of cross-linking of potato starch by 0.05–0.10% POCl3 had no

effect on pancreatin digestion of the gelatinized starch (Janzen, 1969), but

considerable inhibition of starch hydrolysis by pancreatin was displayed at

higher levels of POCl3 treatment (0.5–1.5%). Digestibility of raw or gelati-

nized starch to pancreatic a-amylase was very slightly reduced (1–3%) by low

level cross-linking (�0.0825% POCl3) of lentil, faba bean and field pea

starches (Hoover & Sosulski, 1986). Using epichlorohydrin as a cross-linking

50 Resistant Starch

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agent at 0.1%, 0.3% and 0.5% levels, starch from two Croatian wheat varieties

(Ackar et al., 2010) yielded low levels of resistant starch (0.31–2.25%)

compared to the respective native wheat starches (0.48–0.58%). By compari-

son, 0.06–0.12% STMP treatment of waxy maize starch slightly reduced the

four-hour percent hydrolysis (13.0–13.7%) of the gelatinized starch by hog

pancreatic a-amylase compared to the gelatinized unmodified waxy maize

starch (15.8%) (Hahn & Hood, 1980).

Wheat starch cross-linked with 0.1% POCl3 or 0.3% epichlorohydrin did

not generate significant RS when assayed for total dietary fibre by AOAC

Method 991.43 (Woo & Seib, 2002). However, reaction with 1–2% POCl3 or

1–2% epichlorohydrin yielded 52.7–85.6% total dietary fibre and 57.4–75.8%

total dietary fibre, respectively. The cross-linking conditions were 33% starch

solids, 15% Na2SO4, pH 11.5, reaction temperature of 25 �C, and one hour

reaction time. The use of 12% of a 99 : 1 blend of STMP and sodium

tripolyphosphate (STPP) as modifying agents at approximately the same

reaction conditions as above, except for 10% Na2SO4, reaction temperature of

45 �C, and three hours reaction time, resulted in a phosphorylated cross-linked

RS4 product with low swelling power (�3), 0.32% phosphorus and 75.7%

total dietary fibre. At a 10% level of 99: 1 ratio of STMP/STPP, the level of

phosphorus is 0.32% and total dietary fibre is 75.6%, but other ratios (25 : 75,

50 : 50, or 75 : 25) of STMP/STPP generated lower phosphorus incorporation

(0.13–0.29%) and diminished total dietary fibre (21.6–63.7%).

General cross-linking conditions of 0–20% Na2SO4, 10–19% STMP/STPP

(99 : 1 ratio), pH 11.5–12.3, reaction temperature of 25–70 �C and reaction

time of 0.5–12 hours were utilized for preparation of different phosphorylated

cross-linked RS4 wheat starches with 0.32–0.35% phosphorus and

75.7–88.1% total dietary fibre. Phosphorylated cross-linked RS4 starches

from normal wheat, waxy wheat, normal corn, waxy corn and potato showed

0.32%, 0.32%, 0.34%, 0.33% and 0.32% phosphorus and 76%, 80%, 35%,

58% and 73% total dietary fibre, respectively.

Four phosphorylated cross-linked RS4 wheat starches with phosphorus

contents of 0.13–0.38% and total dietary fibre levels of 14.0–93.4% (Woo &

Seib, 2002) were analyzed for the amount of RS by the Englyst method

(Englyst et al., 1992). The resulting RS levels by this method, before and after

gelatinization of the samples, correlated directly with total dietary fibre using

AOAC Method 991.43. Actual RS values ranged from 35.5–66.1% for the

ungelatinized samples and 2.6–15.2% for the gelatinized samples. Corn starch

cross-linked with 4%, 8%, and 12% of a 99 : 1 mixture of STMP/STPP

introduced 0.14–0.37% phosphorus to the granules, which was directly

proportional (R2¼ 0.9971) to the amount of RS (24.5–81.6%), as measured

by AOAC Method 991.43 (Chung et al., 2004).


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Woo et al. (2006) reported on a pregelatinized, phosphorylated, cross-

linked RS4 starches from wheat, potato and tapioca that exhibited resistance

to a-amylase digestion, possessed fat replacement properties and demon-

strated stability to freeze-thaw processing. This particular pregelatinized RS4

starch was formulated in a number of reduced-fat foods, namely bakery

products, instant mashed potato, sausages, salad dressings, desserts, ice

cream, yogurt and cr�eme filling.

Although Kerr & Cleveland (1957) pioneered the cross-linking of starch at

alkaline pH using STMP, its combination with small levels of STPP during the

reaction formed the basis for patenting a phosphorylated cross-linked food-

grade starch resistant to a-amylase and the method of preparing the same

product (Seib & Woo, 1999). Analysis of phosphodextrins from a-amylase

and glucoamylase digestion of phosphorylated cross-linked RS4 wheat starch

by proton-decoupled 31P NMR (Sang et al., 2007) showed four types of

phosphate esters: cyclic-monostarch monophosphate (cyclic-MSMP), mono-

starch monophosphate (MSMP), monostarch diphosphate (MSDP), and dis-

tarch monophosphate (DSMP), with signals centred at d 15.9 ppm, d 3–5 ppm,

d�5 and�10 ppm, and d�1 to1 ppm, respectively. Phosphorylation of wheat

starch with STMP/STPP produced �37% MSMP and 63% DSMP, whereas

the use of POCl3 changed the proportion to �20% MSMP and 80% DSMP

(Sang et al., 2007).

Starch hydroxyls have ionization constants between pKa 12.5–13.0 at

45 �C (Lammers et al., 1993). Less than 1% of hydroxyl groups is ionized at

pH 10.5, and about 5–10% are ionized at pH 11.5. Depending on the pH used

during the reaction with STMP, the ratio of DSMP/MSMP can vary from

41 : 22 for pH 10.5 to approximately 65 : 35 for higher pH (11.5–12.5). The

lower-reaction pH of 10.5 also produced 18% MSDP and 19% cyclic-MSMP.

The proposed reaction mechanism between starch and STMP at alkaline

pH involves the initial attack by a starch alcoholate ion, resulting in ring-

opening of STMP and formation of a monostarch triphosphate intermediate.

This is followed by the reaction of another starch alcoholate ion with the

intermediate, forming DSMP and pyrophosphate. The formation of MSDP

occurs by losing the g-phosphoryl group on the monostarch triphosphate by

the peeling mechanism. Cyclic-MSMP and MSDP are reactive at high

alkalinity (pH 11.5 for 3 hours), which causes the disappearance of cyclic-

MSMP and the reduction of MSDP from 17% to 11%. The DSMP content of

phosphorylated cross-linked RS4 wheat starch correlated positively (r¼ 0.96;

P¼ 0.02) with RS content (as determined by the method of Englyst et al.

(1992)) and with total dietary fibre content (r¼ 0.90; P¼ 0.05) as assayed by

AOAC Method 991.43.

52 Resistant Starch

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Sang et al. (2010) reported a phosphorylated cross-linked RS4wheat starch

(0.37% phosphorus, 88.8% total dietary fibre) exposed to NaOH at pH 9–11

and 40 �C for four hours that did not significantly change the phosphorus

content (0.35–0.37%), even with the elevated pH treatment. A much higher

alkalinity of pH 12 at the above time-temperature combination reduced the

phosphorus content to 0.29%, which indicates that about 22% of covalently-

bound phosphate was removed from the starch. In addition, cyclic-MSMP and

MSDP disappeared, as confirmed by the absence of 31P NMR signals, which

suggests the instability of those two types of phosphate esters at pH 12.

However, the cross-linked phosphate ester, DSMP, increased to �18% after

exposure at pH 12 and 40 �C for four hours, indicating that new DSMP was

formed from the reaction of MSDP or cyclic-MSMP with hydroxyl groups on

another starch chain. Total dietary fibre content was little changed (88.8% vs.

86.5%) upon exposure to pH 12, even though the total phosphorus decreased

by 22%. The 18% increase of cross-linked phosphate esters (DSMP) com-

pensated for the 22% loss of total phosphorus. The integrated NMR intensities

of DSMP andMSMP indicated that they contained 0.17% phosphorus (or 46%

of total phosphorus) and 0.077% phosphorus (or 21% of total phosphorus),

respectively.

Starch citrate is the nomenclature used in the literature for the cross-linked

product derived from high-temperature treatment of a low-moisture mixture

of starch and citric acid, a six-carbon tricarboxylic acid. The reaction

mechanism involves dehydration of citric acid to anhydride form, and this

functional group consequently forms cross-linking bonds with starch mole-

cules. Klaushofer et al. (1978) developed a cross-linked starch using citric

acid as the primary modifying agent. The process involved drying to 5–20%

moisture an aqueous mixture of starch and citric acid (5–40%, starch basis)

and heating for 1–5 hours at 110–140 �C to yield starch citrate. This product

exhibited decreased in vitro amylase digestibility with an increasing degree of

esterification. In addition, the esterified starch showed improved stability to

shear and freeze-thaw processing conditions.

Xie & Liu (2004) reported that for a corn starch/citric acid weight ratio of

5 : 2, pH 3.5 and a reaction temperature range of 120–150 �C for 3–9 hours, the

DS varied from 0.09–0.12 and the level of RS ranged from 41.1–78.8%. An

improvement of RS content to 92.9% was achieved when a reaction tempera-

ture of 150 �C was employed. On the other hand, Wepner et al. (1999)

obtained 45.9–57.5% RS from starch citrates prepared from potato, pea, corn,

and wheat starches using the following reaction conditions: starch/citric acid

weight ratio of 5 : 2, pH 3.5 and five hours at 140 �C. The DS expressed as

percent esterified citric acid ranged from 12.2–14.4%.


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Citric acid-treated starch containing slowly digestible and resistant starch

fractions was described by Shin et al. (2007). The preparation procedure

consisted of a two-step process of autoclaving (121 �C for 30 minutes) an

aqueous mixture of starch-citric acid, followed by heat treatment as high as

130 �C for up to 24 hours. The resulting product has a dextrose equivalent

(DE) of 10.2, DS of 0.027, cold water solubility of 55.2%, slowly digestible

and resistant starch fractions of 54.1%, and low blood glucose response

in mice.

Other researchers reacted glutaric acid, a five-carbon dicarboxylic acid,

with adlay starch in preparing cross-linked RS4 resistant starch and charac-

terizing its properties (Kim et al., 2008). Starch glutarate with high RS content

of 65–66% was prepared from adlay starch using the following reaction

conditions: 30–40% glutaric acid (starch basis), 115–130 �C, and 6–7.5 hoursof reaction time. The modified product displayed a carbonyl absorption band

at 1730 cm�1 by FT-IR and 173 ppm and 32 ppm CP-MAS 13C NMR peaks

assigned to carbonyl and methylene groups, respectively, suggesting cross-

linking between starch chains by esterification. It was observed that formation

of b-1,6-glycosidic bonds may have also occurred during the preparation of

starch glutarate.

3.3.2 Substituted RS4 starches

Food-grade substituted starches used for technological effects are normally

treated with appropriate modifying agents (for example, acetic anhydride or

propylene oxide) to yield a DS or a molar substitution (MS) of �0.1 (Mason,

2009). These esterified or etherified starches have the general properties of

increased swelling, elevated viscosity and enhanced clarity, and their pastes

have improved stability to room temperature, as well as refrigerated or freezer

storage.

Pancreatin digestibility of gelatinized hydroxypropylated potato starch

decreased with increasing DS (Leegwater & Luten, 1971). A logarithmic plot

of DS (0.02–0.45) and reducing power (2.6–61.0mg glucose per mmol chain

unit) demonstrated that the digestibility decreased exponentially with increas-

ing DS. In a related observation, Klaushofer et al. (1978) reported declining in

vitro amylase digestibility with an increasing degree of esterification with

citrate. Furthermore, an increasing degree of hydroxypropyl substitution

among cereal starches resulted in a diminished in vitro enzymatic hydrolysis

(Azemi & Wooton, 1984).

Hahn & Hood (1980) showed that gelatinized hydroxypropylated waxy

maize starch (MS¼ 0.095–0.131) was digested to 11.4% after four hours of

hydrolysis with hog pancreatic a-amylase, compared to 15.8% hydrolysis of

54 Resistant Starch

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the gelatinized unmodified waxy maize starch. In another study, the digest-

ibility of gelatinized wheat starch by porcine pancreatic a-amylase was

reduced by 13.3–23.4% by substitution with hydroxypropyl (DS¼ 0.06) or

acetyl groups (DS¼ 0.07), while cross-linking with phosphate (0.05% POCl3)

had a minor lowering effect (2.5%) on its digestibility (Wooton & Chaudhry,

1979). Etherification with hydroxypropyl groups lowers the digestibility of

wheat starch to a larger extent (23.7–25.2%) than esterification with acetyl

groups (10.8–13.3%), most likely due to the larger influence of the more bulky

hydroxypropyl group on enzyme digestion.

The relative degree of hydrolysis to hog pancreatic a-amylase

(0.0175–0.30% enzyme concentration based on starch) was 75.3–80.1%

for gelatinized tapioca hydroxypropyl distarch phosphate (1.6% hydrox-

ypropyl) and 50.6–58.2% for gelatinized hydroxypropylated tapioca starch

(4.2% hydroxypropyl), compared to 100% for gelatinized unmodified

tapioca starch (Conway & Hood, 1976). These digestibility values are

comparable to other studies conducted on hydroxypropylated starches by

Leegwater & Luten (1971) and by Hood & Arneson (1976).

Highly hydroxypropylated (DS¼ 0.12), lightly cross-linked (0.000085%

POCl3) waxy starch showed decreased digestibility (34.4%) to a-amylase/

glucoamylase compared to unmodified waxy starch (98.0%) (Wolf et al.,

1999). Moderately hydroxypropylated (DS¼ 0.07), moderately cross-linked

(0.00037% POCl3) dullwaxy starch displayed decreased digestibility (68.7%)

compared to unmodified dull waxy starch (90.6%).

Due to their beneficial health effects, acylated starches that possess much

higher DS compared to traditional food-grade modified starches were sug-

gested for possible use in food products (Annison et al., 2003; Bajka et al.,

2010). Acylated starches as a form of substituted RS4 starch was borne out of

a strategy to deliver short chain fatty acids directly to the large bowel (Annison

et al., 1995). The 2-, 3- or 4-carbon short-chain fatty acids represented by

acetic acid, propionic acid or butyric acid, respectively, can be attached to

starch by an esterification process. Annison et al. (2003) reported on the

preparation of these esters with a DS of approximately 0.20 by reacting 600 g

starch with 115ml, 180ml or 230ml of acetic anhydride, propionic anhydride

or butyric anhydride, respectively.

Butyrate esters of corn starch tended to increase starch resistance to

enzymic hydrolysis in vitro and to intestinal amylolysis in vivo (Annison

et al., 1995, 2003). Butyrate is released in the large bowel by the action of

microbial esterases and lipases, leaving the starch backbone available for

fermentation (Bajka et al., 2006, 2007). A butyrylated starch product with a

DS of approximately 0.3 was exceedingly resistant to amylolysis, but was still

capable of liberating butyrate when exposed to faecal microflora (Bajka et al.,


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2007). Less butyrate is released when the DS is greater than 0.3, which may be

explained by decreased solubility or increased steric obstruction to the action

of microbial esterases.

Waxy maize starch esterified with 3% octenyl succinic anhydride showed

28.3% slowly digestible starch, which rose to 42.8% after the succinylated

starch was heat-moisture treated at 120 �C for four hours and 10% moisture

(He et al., 2008). Cooked 1-octenyl succinylated corn starch (DS¼ 0.07;

DE� 3) decreased the extent of 15-hour in vitro hydrolysis (70%) by

a-amylase/amyloglucosidase (Wolf et al., 2001) compared to cooked,

unmodified corn starch (99.9%). Cooking of butyrylated high-amylose

corn starch increased in vitro a-amylase/amyloglucosidase hydrolysis from

6% to 43% (Bajka et al., 2006).

3.3.3 Pyrodextrinized RS4 starches

Pyrodextrins are conventionally produced by treatment of granular native

starch at low moisture contents and elevated temperatures with varying

amounts of mineral acids and, sometimes, carboxylic acids (Wurzburg,

1986; Tomasik et al., 1989; Wang et al., 2001; Laurentin et al., 2003; Huber

& BeMiller, 2010). These conditions destroy the original starch backbone

structure and promote hydrolysis, transglucosidation and repolymerization.

As a result, pyrodextrins have low molecular weights, possess atypical

glycosidic linkages (i.e. (1! 2)-, (1! 3)-glycosidic bonds) and contain

new branched structures.

Using dextrinization conditions of pH 2.5–2.6, a temperature of 170 �C,and a three-hour reaction time, Wang et al. (2001) demonstrated, by reducing

sugar analysis, that hydrolysis occurred during the first hour, followed by

transglucosidation and repolymerization in the remaining two hours. This

result agreed with the low content of enzyme resistant dextrins during the first

hour of pyrodextrinization, followed by a rapid increase of enzyme-resistant

dextrins in the next two hours.

Pyrodextrinization, followed by amylase treatment, was the basis for a

number of patents to produce soluble dietary fibres whose nomenclature may

fall under different names, such as indigestible dextrins, resistant dextrins,

resistant maltodextrins or digestion-resistant maltodextrins (Ohkuma et al.,

1994, 1995, 1997). A product of this type dissolves into a clear and stable

aqueous solution with low viscosity. It has a DE of 10–12, an average

molecular weight of 2000Da, and its structure is composed of (1! 2)-,

(1! 3)-, (1! 4)-, and (1! 6)-glycosidic linkages and levoglucosan

(Ohkuma & Wakabayashi, 2001; Okuma & Kishimoto, 2004). It is approxi-

mately 90% indigestible, with a low caloric count (0.5 kcal/g). Resistant

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maltodextrins that are available commercially vary in total dietary fibre

content, ranging from 52–90%, and they are being offered either as a

hydrogenated version, in agglomerated form, in admixture with honey solids

and stevia glycosides, or in liquid corn syrup version (75% solids).

Another manufacturing process for resistant dextrins involves controlled

dextrinization, wherein the starch undergoes a degree of hydrolysis followed

by repolymerization (Lefranc-Millot et al., 2010). This process converts

starch into dietary fibre through the formation of non-digestible glycosidic

bonds that deter cleavage by amylase enzymes. When analyzed by AOAC

Method 2001.03, the resistant dextrin obtained in this process contains 85%

total dietary fibre, which consists of 50% insoluble fibre in ethanol and 35%

resistant oligosaccharides. In another study, lightly, moderately and highly

converted dextrins from common corn starch displayed digestibilities of

90.1%, 84.2% and 63.8%, respectively, when treated with a mixture of

a-amylase and glucoamylase, whereas raw common corn starch has 97.9%

digestibility (Wolf et al., 1999).

Formation of enzyme-resistant fractions during pyrodextrinization was

shown to be affected by the botanical source of starch (Laurentin et al., 2003).

These researchers also revealed that granule morphology of native starch was

similar to that of pyrodextrinized starch, and that the latter exhibited no

endotherm by differential scanning calorimetry (DSC). Extensive

depolymerization of pyrodextrinized starches occurred as evident in the

gel filtration profile, and the amount of starch hydrolysable by the combined

action of Termamyl a-amylase and amyloglucosidase was significantly

decreased.

3.4 PHYSICOCHEMICAL PROPERTIES AFFECTINGFUNCTIONALITY

The chemical name of a phosphorylated cross-linked RS4 starch produced by

treatment with a mixture of STMP and STPP at a 99 : 1 ratio (Seib & Woo,

1999) is phosphated distarch phosphate (E-Number 1413 and CAS No.

977043-58-5). The use of these two phosphate-modifying agents is regulated

to yield a food-grade product with nomore than 0.4% phosphorus, as specified

in the section on Food-StarchModified of the US Code of Federal Regulations

Title 21 Part 172.892 (Code of Federal Regulations, 2010). Commercial-scale

production yielded a product that delivered a minimum of 85% (dry basis)

total dietary fibre with an average of �94% (dry basis) total dietary fibre by

AOAC Method 991.43 (Woo et al., 2009). The fibre exists primarily as

insoluble fibre. A phosphorylated cross-linked RS4 wheat starch was


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described in a US patent publication to contain 93.4% total dietary fibre and

0.38% phosphorus (Seib & Woo, 1999).

Phosphorylated cross-linked RS4 starches from wheat, corn, potato and

rice are practically insoluble in dimethyl sulphoxide or 1M KOH (Woo &

Seib, 2002; Shin & Seib, 2004). Scanning electron microscopy showed the

same shape and smooth surface as their parent starches (Seib & Woo, 1999).

Pasting curves at 8% starch solids did not rise above the baseline when heated

from 30 �C to 95 �C. Wide-angle x-ray diffraction showed phosphorylated

cross-linked RS4 starches from cereal, and potato sources gave A- and B-type

polymorphic crystal patterns, respectively.

When analyzed by DSC, the transition temperatures of phosphorylated

cross-linked RS4 potato starch were slightly affected. In contrast, the other

cross-linked RS4 starches from normal wheat, waxy wheat, normal corn

and waxy corn gave elevated To, Tp, and Tc by 4.3–8.4 �C, 5.4–8.3 �C, and3.6–10 �C, respectively (Woo & Seib, 2002). Enthalpy of gelatinization

tended to be lower for all the RS4 starches, except for RS4 potato starch.

It was surmised that annealing of starch occurred during exposure to the cross-

linking reaction conditions, and that cross-linking could inhibit cooperative

melting of crystals in starch granules (Woo & Seib, 2002; Jacobs et al., 1995).

No significant differences in DSC transition temperatures and enthalpies of

gelatinization were found after a phosphorylated cross-linked RS4 wheat

starch was held at pH 9.0–12.0 for four hours at 40 �C (Sang et al., 2010). This

alkaline pH treatment did not alter its typical A-type x-ray diffraction pattern

or its degree of crystallinity.

The phosphorus content of phosphorylated cross-linked RS4 corn starch

modified with 4–12% of a 99 : 1 blend of STMP/STPP was inversely

proportional (R2¼�0.9537) to the swelling volume of the cross-linked

product (Chung et al., 2004). Cross-linking has the added effect of increasing

the glass transition temperature of corn starch in excess water by nearly 1 �C,which was explained as a reduction in chain mobility, such that a higher

temperature (Tg) is required to induce movement. On the other hand, the glass

transition temperature of the cross-linked starches measured at 15% moisture

decreased by �1 �C, which was attributed to internal plasticization of starch

by the ionic phosphate groups. Furthermore, the retrogradation enthalpy after

one week of storage of the gelatinized starches (67% moisture) at 4 �C was

decreased by cross-linking.

Cross-linking of banana starch with 11.9% STMP and 0.1% STPP raised

the DSC peak temperature of gelatinization to 86.6 �C, compared to 79.2 �Cfor the parent native banana starch, while the enthalpy of gelatinization

decreased to 9.36 J/g from 25.1 J/g for the parent native banana starch

(Aparicio-Saguilan et al., 2008). Autoclaving at 121 �C for one hour, followed

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by storing at 4 �C for 24 hours, decreased the RS content (total dietary fibre

assay) of phosphorylated cross-linked banana starch from 94.68% to 85.35%.

As a result of autoclaving and cooling treatments, the peak temperature and

enthalpy of gelatinization decreased to 68.5 �C and 1.5 J/g, respectively.

When a phosphorylated cross-linked RS4 wheat starch was heated in a

cooking viscometer (8% solids) from 30 �C to 95 �C, no pasting curve (i.e. flatline) was generated, which is indicative of the highly restricted swelling of the

cross-linked starch granules (Woo & Seib, 2002; Xie & Liu, 2004). The

granules remained intact after heating, and it was observed that disintegrated

or convoluted granules were not evident, and neither were granule ghosts.

DSC analysis of phosphorylated cross-linked RS4 wheat starch demonstrated

elevated initial, peak, and conclusion temperatures (4.3–10.5 �C higher), but a

slight reduction in enthalpy of gelatinization (0.9 J/g lower) compared to the

parent native wheat starch.

When heated from 35 �C to 60 �C in excess water, the granules of a

commercial phosphorylated cross-linked RS4 wheat starch maintained their

structures essentially unchanged (Ratnayake & Jackson, 2008). Above 65 �Cand up to 85 �C, the granules became increasingly swollen. Coinciding with

this observation, the DSC enthalpies of gelatinization did not change during

the 35–60 �C treatment, but gradually disappeared between 60 �C and 85 �C.X-ray crystallinity gradually decreased within the same temperature range of

60–85 �C, but the A-type polymorph was preserved up to 70 �C. Consistentwith previous studies (Seib & Woo, 1999; Woo & Seib, 2002), Ratnayake &

Jackson (2008) found that phosphorylated cross-linked RS4 wheat starch did

not completely dissolve in 90% dimethyl sulphoxide.

A phosphorylated cross-linked RS4 wheat starch with 72.9% total dietary

fibre exhibited a low swelling power of 2.8 g/g and low solubility in water of

0.5% at 95 �C (Woo & Seib, 2002). Compared to other cereal starches, it

displayed similar water vapour sorption and desorption isotherms at 25 �C and

at water activities below 0.8 (Shin et al., 2003).

Lefranc-Millot et al. (2010) described a resistant pyrodextrin that has a

clean, neutral taste, possesses no sweetness and is compatible with sugar-free

claims. The commercial product dissolves rapidly in aqueous systems and has

limited impact on viscosity. It is stable to high temperature, variable pH and

high shear processing. The resistant dextrin is officially recognized as a

soluble dietary fibre, with suggested ingredient labelling of ‘dextrins’ in food

product packages.

Consistent with heavily cross-linked starches, corn starch citrates resisted

granular swelling as exhibited by the flat RVA pasting curve (i.e. 0 RVU) when

heated from 50 �C to 95 �C at 8% starch solids (Xie & Liu, 2004). Heating

starch citrates in water (7% solids) at 100 �C for 30 minutes resulted in


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9.8–16.8% loss in RS content, indicating intermediate thermal stability of this

product. The DSC enthalpy of gelatinization tended to decrease in starch

citrates (0.4–3.0 J/g), compared to the native corn starch (6.7–15.6 J/g).

Analysis by DSC demonstrated that adlay starch glutarates with RS

content of 30–66% exhibited lower To (46.0–49.9 �C), Tp (60.5–65.6 �C),Tc (73.1–73.9

�C), andDH (5.7–9.5 J/g) compared to control starch (To, 60�C;

Tp, 71.3�C; Tc, 82.6

�C; D H, 13.3 J/g), presumably because of the disruption

of molecular (double helical) and crystalline orders during the preparation of

the glutarates (Kim et al., 2008). Hot water solubility at 80 �C of the glutarate

derivatives was drastically reduced to 0.71–1.52%, compared to 81.2% for the

control starch. When subjected to a-amylase and amyloglucosidase treat-

ments, gelatinized starch glutarate lost its granular shape and its granules

cohered together. Its good stability to heat treatment in excess water was

demonstrated when the RS content before heating of starch glutarate was

shown to be similar to that after heating.

3.5 PHYSIOLOGICAL RESPONSES AND HEALTHBENEFITS

Dietary fibre sources, including RS, promote a number of physiological

benefits in humans, which include, but are not limited to, the following:

decreased intestinal transit time; enhanced satiety; reduced postprandial blood

glucose and/or insulin levels; diminished blood total and/or low-density

lipoprotein cholesterol concentrations; and fermentability by colonic micro-

biota to give short-chain fatty acids (SCFA) (Baghurst et al., 1996; Topping &

Clifton, 2001; Nugent, 2005; Topping, 2007;Witwer, 2008; Buttriss & Stokes,

2008; Bird et al., 2009).

Different types of RS can elicit significantly different glycemic responses,

as demonstrated by a comparison between RS2 high-amylose corn starch

and phosphorylated cross-linked RS4 wheat starch in human subjects

(Haub et al., 2010). The glucose response and the incremental area under

the curve of phosphorylated cross-linked RS4 wheat starch were signifi-

cantly decreased, compared with RS2 high-amylose corn starch and dex-

trose (control).

Aqueous dispersions of two RS4 potato starches (38 grams in 296ml of

water), when consumed as a drink (Haub et al., 2012), elicited a significantly

decreased glycemic response compared to dextrose (control). There were no

blood glucose response differences and no satiety effects when the same doses

of RS4 potato starches were added to dextrose and compared with dextrose

alone. Rice kernels coated with RS4 wheat starch using agar and locust bean

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gum had lower in vitro starch digestibility, a decreased glucose response in

rats and a slower rate of blood glucose decrease, compared to uncoated rice

and rice mixed with RS4 wheat starch (Choi et al., 2010).

Using the glycemic index protocol, nutritional bars formulated with 34

weight percent of phosphorylated cross-linked RS4 wheat starch displayed

attenuated postprandial blood glucose and insulin levels, when compared to a

control glucose drink and to another nutritional bar in which the above RS4

wheat starch was replaced by an equivalent amount of puffed wheat

(Al-Tamimi et al., 2010). Incremental areas under the glucose curves were

140, 84 and 28mmol/l� 2 hour for the glucose control drink, puffed wheat bar

and cross-linked RS4 bar, respectively. The corresponding incremental areas

under the insulin curves were 17 575, 8758, and 3659 pM� 2 hour, respec-

tively. The results are noteworthy, as the bar formulas contain high levels of

glycemic carbohydrates, in the form of 11% brown sugar and 20% corn syrup.

Other researchers compared the properties in vivo of phosphorylated cross-

linked RS4 wheat starch and RS2 high-amylose corn starch in order to gauge

their impact on gastrointestinal microbiota composition and metabolism in

human volunteers (Martinez et al., 2010). During the three-week feeding

period, both types of resistant starch were well tolerated by the subjects when

fed daily with 100 grams of snack crackers (containing 33 grams of dietary

fibre contributed by the resistant starch ingredient). Pyrosequencing of faecal

samples demonstrated that, at the phyla level, RS4 crackers significantly

decreased Firmicutes and increased Bacteroidetes and Actinobacteria. These

changes were associated with a decrease in the family Ruminococcaceae and

increases in the genera Parabacteroidetes and Bifidobacterium.

Denaturing gradient gel electrophoresis revealed that RS4 crackers induced

a swift and reversible increase in band intensity of Bifidobacterium adoles-

centis, whereas RS2 crackers caused a gradual rise. Quantitative enumeration

of bifidobacteria by qRT-PCR confirmed the significant increase in cell

numbers during consumption of RS4 and RS2 crackers, compared to the

control crackers (no resistant starch). The total cell numbers of bifidobacteria

increased more than three-fold with RS4 crackers, while RS2 crackers

doubled the cell numbers.

In vitro digestion of phosphorylated cross-linked RS4 wheat starch and

phosphorylated cross-linked RS4 potato starch by successive treatments with

pepsin and pancreatin-bile yielded 82% and 74% of indigestible residues,

respectively (Thompson et al., 2011). Incubation of these indigestible residues

with fresh human faecal inoculum produced gases that increased linearly over

the 24-hour fermentation period, with the two RS4 starches exhibiting similar

gas production rates as well as similar rates of production of total short-chain

fatty acids. Acetic acid was the major SCFA produced, along with relatively


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higher amounts of butyric acid than propionic acid. The fractional molar ratios

of the above acids (acetic : propionic : butyric) are 0.586 : 0.186 : 0.228 and

0.577 : 0.200 : 0.223 for RS4 wheat starch and RS4 potato starch, respectively.

Caloric contribution was calculated to be one-third lower for the two RS4

starches, compared to unmodified (non-resistant) starch.

In an animal feeding trial, hamsters on a phosphorylated cross-linked RS4

wheat starch diet consumed less feed and gained less weight than those on a

cellulose diet (Seib &Woo, 1999). Total serum cholesterol in the animals was

not significantly different between the two diets, but the concentration of high-

density lipoprotein cholesterol was significantly higher in the RS4 diet.

Consequently, the calculated levels of low- and very low-density lipoprotein

cholesterols were less in the serum of animals consuming the RS4 diet than the

cellulose diet. Analysis of fermentation products from the caecum of hamsters

showed elevated levels of SCFA (in particular butyric acid) in the RS4 diet,

compared to the cellulose diet.

In mice fed for six weeks with a high-fat diet, supplemental addition at a

15% level of phosphorylated cross-linked RS4 corn starch and phosphorylated

cross-linked RS4 high-amylose corn starch lowered body weight gain as well

as total lipid, triglyceride and total cholesterol in the serum and liver,

compared to the corresponding unmodified starches (Lee et al., 2012).

Rats fed for four weeks with a diet containing 5% phosphorylated cross-

linked RS4 corn starch (92.6% total dietary fibre) exhibited lower serum levels

of total cholesterol (12.6% decrease) and low density lipoprotein plus very

low density lipoprotein cholesterol (24.3% decrease) than rats fed with a diet

containing 5% cellulose (Song et al., 2010). In addition, this RS4 corn starch

can potentially ameliorate allergic inflammation in the mesenteric lymph

nodes of rats, because of its impact on elevating serum immunoglobulin A

levels, as well as the CD4þ T cell population and the ratio of CD4þ/CD8þ T

cells.

Annison et al. (2003) summarized the strategies to elevate SCFA in the

large bowel as follows:

a) Increase the consumption of foods high in RS content.

b) Consume SCFA directly in foods or beverages.

c) Consume foods that contain starches esterified with specific SCFA.

The rat feeding study by these researchers confirmed that starches esterified

with specific SCFA through reaction with acetic, propionic or butyric anhy-

dride resisted digestion in the small intestines and delivered those SCFAs into

the large bowel, where they were released by the action of the microflora. The

selective increase of individual SCFAwas observed even only after three days,

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which indicates that the rat microflora can readily adapt. Esterified starches

with a DS of around 0.20–0.25 have the potential to improve the nutritional

properties of foods through delivery of SCFA directly to the large bowel.

Ingestion by ileostomists of starches esterified with acetate, propionate or

butyrate with DS of 0.23–0.25 resulted in the recovery of 73–76% of the

esterified acid at the terminal ileum, which suggests that SCFA delivery to the

large bowel could occur in humans with an intact gastrointestinal tract (Clarke

et al., 2007). Free faecal butyrate concentrations were increased in human

subjects after consumption of butyrylated high-amylose corn starch, and about

27% of total esterified butyrate was absorbed in the small intestine, 15.8%was

recovered in the faeces, and about 57.2% was released in the large bowel

(Clarke et al., 2011a). The population of Parabacteroides distasonis was also

increased by the above-mentioned butyrylated starch derivative.

Reduction in DNA single-strand breaks was twice that in rats fed butyry-

lated high-amylose corn starch compared to unesterified high-amylose corn

starch, which was interpreted as the greater ability of the butyrylated starch to

deliver butyrate to the large bowel, thereby raising the concentration of this

particular SCFA (Bajka et al., 2008). High-protein diets increased colonocyte

DNA single-strand breaks in rats, but this effect was opposed by inclusion in

the diet of butyrylated high-amylose corn starch (Conlon et al., 2012).

Butyrylated high-amylose corn starch protects colonocytes in rats from

genetic damage by high dietary protein (Bajka et al., 2008), which may be

explained by increased caecal butyrate pools.

Bajka et al. (2006) studied the effects of feeding high-amylose corn starch

and butyrylated high-amylose corn starch in rats and the effect of cooking on

resistance to in vitro and in vivo amylolysis. Cooking of butyrylated high-

amylose corn starch increased in vitro a-amylase/amyloglucosidase hydroly-

sis from 6% to 43%. Cooked butyrylated high-amylose corn starch delivered

significantly greater amounts of esterified butyrate in the rats’ large bowel,

compared to raw or cooked high-amylose corn starch. The caecum is the main

site of bacterial fermentation of carbohydrates in rats. By comparison,

ingestion of butyrylated low-amylose corn starch by rats resulted in lower

digesta pH in the large bowel and elevated butyrate concentrations in the

caecal, proximal and distal colon, as well as in the portal vein (Bajka et al.,

2010).

Butyrylated high-amylose corn starch (DS¼ 0.25) affected the faecal

microbiota composition of rats treated with azoxymethane, a carcinogenic

agent (Abell et al., 2011). It increased propionate and butyrate concentrations

in distal colonic digesta and was associated with the appearance of Para-

bacteroidetes distasonis, Lactobacillus gasseri and Phascolarctobacterium

faecium. Following acute exposure to azoxymethane, rats fed with butyrylated


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high-amylose corn starch had higher caecal tissue and digesta weights and

lower pH in the large bowel, compared with rats fed with unmodified low-

amylose and high-amylose corn starches (Clarke et al., 2011b). Morphologi-

cal assessment of the rats’ distal colonic epithelium showed that butyrylation

has a positive impact, as indicated by an elevated apoptotic index. Butyrate

concentrations in hepatic portal plasma, as well as in caecal and proximal and

distal colonic digesta, were higher in azoxymethane-treated rats fed with

butyrylated high-amylose corn starch, compared to non-acylated low-amylose

and high-amylose corn starches (Clarke et al., 2008). This butyrate effect is

associated with reduced tumour incidence, number and size in rats.

Net incremental area under the blood glucose curve (Wolf et al., 2001) was

lower (P< 0.05) after fasting healthy adult subjects consumed 25 g of

1-octenyl succinylated starch (107mmol.min/l) than that for 25 g of glucose

(127mmol.min/l). A blunted glycemic response was displayed by 1-octenyl

succinylated starch, as indicated by the calculated relative glycemic response

of 93.8% compared to that of glucose.

Digestibility in the small intestine of a resistant pyrodextrin (Lefranc-

Millot et al., 2010) is in the range of 8.7–19%, which results in a low glycemic

response (�25) and low insulinemic response (�13). The low insulinemic

response is reported to induce a high satiety feeling and to reduce postprandial

hypoglycaemia. When the resistant pyrodextrin was formulated in pasta,

beverages and biscuits, consumption of these food products displayed low

glycemic responses (Lefranc-Millot et al., 2006).

In a cross-over feeding study, 20 volunteers consuming 300 g rice with

5.7 g of resistant maltodextrins displayed significantly lower postprandial

blood insulin and glucose levels, compared to rice without resistant malto-

dextrins. When 13 volunteers ate a fast-food restaurant meal with 10 g of

resistant maltodextrins, the insulin level at the 30-minute postprandial period

was significantly reduced in comparison to the same meal without resistant

maltodextrins (Gordon, 2007).

Other researchers confirmed in vivo the blunting effect of resistant malto-

dextrins on postprandial blood glucose and insulin levels, coupled with a

decrease in intestinal glucagon (Okuma & Kishimoto, 2004; Wakabayashi

et al., 1992; Tokunaga & Matsuoka, 1999). Co-ingestion of resistant malto-

dextrins with monosaccharides or disaccharides can moderate increases in

postprandial blood glucose levels, resulting in reduced insulin requirements

and preventing fat accumulation (Ohkuma & Wakabayashi, 2001). Among

Type 2 diabetics, 10–20 g of resistant maltodextrins fed at every meal for three

months significantly reduced fasting blood glucose levels, improved glucose

tolerance and decreased HbA1C (Nomura et al., 1992; Fujiwara &Matsuoka,

1995).

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Body fat was significantly reduced when 12 overweight males with

hyperlipidaemia were fed 30 g of resistant maltodextrins each day for three

months (Gordon, 2007). Long-term feeding of diets with added resistant

maltodextrins to patients with hyperlipidaemia had the general effect of

decreased low-density lipoprotein cholesterol and triglycerides, but increased

high-density lipoprotein cholesterol (Okuma & Kishimoto, 2004).

Supplementation of resistant maltodextrins in the diet at the rate of

7.5–15 g/day for three weeks was well tolerated by human subjects and

tended to increase the faecal Bifidobacterium population (Fastinger et al.,

2008). There was also a shift in the molar proportions of SCFA towards

butyrate. In an earlier study, resistant maltodextrins increased faecal bulk and

decreased symptoms of constipation (Satouchi et al., 1993).

Flickinger et al. (1998) determined that a dextrinized corn starch with

mixed glycosidic linkages was hardly digested in the small intestine of ileally

cannulated dogs. This dextrin has an estimated caloric value of 2.2 kJ/g.

Another mixed-linkage resistant dextrin (non-viscous soluble dietary fibre)

described by Lefranc-Millot et al. (2010), and made either from wheat or corn

starch, has a caloric value of 2 kcal/g (Vermorel et al., 2004), which is in

agreement with the above reported caloric content.

3.6 PERFORMANCE IN FOOD AND BEVERAGEPRODUCTS

Product developers and designers have an arsenal of conventional dietary

fibres and resistant starch ingredients to boost the fibre content of food and

beverage products for nutrient and calorie labelling claims (Baghurst et al.,

1996; Erickson, 2005; Topping, 2007; Witwer, 2008; Bird et al., 2009).

Resistant starches from different sources and belonging to RS1–RS5 types are

traditionally white in colour and possess fine particle size and neutral flavour.

However, they have distinct differences in water-holding capacity that con-

note formulation changes because of the impact on water absorption and

processing parameters of bakery foods (Woo et al., 2009).

A number of bakery, pasta, noodle, dairy, snack and confectionery products

were formulated with a phosphorylated cross-linked RS4 wheat starch to

enhance fibre content, lower net available carbohydrates and reduce caloric

counts (Maningat et al., 2005, 2008). A white pan bread (Yeo & Seib, 2009)

containing one part of phosphorylated cross-linked RS4 wheat starch blended

with nine parts of wheat flour had a slightly lower specific volume (5.96 cc/g)

and slightly firmer texture (427.8 g), compared to a native wheat starch

formula (6.07 g/cc; 475.2 g) and a control wheat flour formula (6.28 g/cc;


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406.9 g). In the same study, sugar-snap cookies were used to compare the

performance of phosphorylated cross-linked RS4 wheat starch with RS2

potato starch. Both starches produced cookies with top grain and spread

factors comparable to the control flour. However, cookies made with RS4

wheat starch exhibited similar snapping force, but RS2 potato starch demon-

strated significantly lower snapping force when compared to the control

cookie.

In a related study, Erickson (2005) formulated an oatmeal chip cookie by

total replacement of cookie flour with RS4 resistant starch. The usage level of

12.78% RS4 resistant starch in the cookie formula delivered a ‘good source of

fibre’ claim (3 g of dietary fibre per 30 g serving size).

High-protein, high-fibre white or whole wheat bread doughs formulated

with 11.6% of phosphorylated cross-linked RS4 wheat starch (based on total

formula weight) had 4–14% higher water absorption, 3–5 minute shorter

mixing time, 17–23 minute shorter proof time, and a baking time about four

minutes longer than the respective control doughs (Maningat et al., 2005).

The high-protein, high-fibre breads displayed greater loaf volume (260–325

cc higher) and higher moisture (3.2–4.6% higher), protein (6.2–9.0%

higher), and dietary fibre (12.4–15.7% higher), along with a �27% average

reduction in calories (by calculation), compared to the respective control

doughs.

Flour tortillas formulated with 5%, 10%, and 15% of phosphorylated cross-

linked RS4 wheat starch generated doughs that were characteristically soft,

extensible and easy to spread, with minimal shrink-back after pressing

(Alviola et al., 2010). The diameters of tortillas containing 10% and 15%

RS4wheat starch were significantly larger (P< 0.05), but the thickness tended

to decrease, compared to the control tortillas. The calculated specific volume

tended to increase as the dosage of RS increased. Flour tortillas with 15% RS

wheat starch had 14.3% dietary fibre and produced significantly higher

(P< 0.05) overall acceptability scores than the control tortillas (dietary

fibre¼ 2.8%).

A pregelatinized version of phosphorylated cross-linked RS4 resistant

wheat starch showed a significant (P< 0.001) and linear (R2¼ 0.97) trend of

increasing flour tortilla weight with an increased level of RS substitution

(Alviola et al., 2010). This increased water requirement results in competition

with gluten for water yielding doughs that were less extensible (resisted

spreading). The resulting tortillas were less puffed and denser than the control

tortillas, but had significantly increased tortilla weight by up to 6.2% and

dietary fibre by 13.6% at a 15% level of substitution.

The addition of RS2 and RS4 high-amylose corn starches in pasta at

2.5–10.0% levels significantly decreased water absorption and swelling index,

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compared to control pasta (Bustos et al., 2011). As the level of RS4 starch

increases, the hardness of pasta tends to increase, but springiness, cohesive-

ness and chewiness tend to decrease.

Corn starch cross-linked with 4–8% of a 99 : 1 mixture of STMP and STPP

produced fried batters with improved crispiness and hardness and reduced oil

uptake, as indicated by the higher TA.XT2 peak number and peak force, along

with lower oil content compared to unmodified corn starch (Han et al., 2007).

The phosphorylated cross-linked RS4 corn starch may have formed a rigid

matrix in the fried batter that increased batter hardness and inhibited oil

absorption during frying.

To measure the textural contribution and test the integrity or survivability

of phosphorylated cross-linked RS4 wheat starch under adverse processing

conditions, it was subjected to a Wenger TX-57 twin-screw extrusion process

during the preparation of a ring-shaped breakfast cereal containing 0%, 5%,

10%, 15% and 20% of the RS4 wheat starch (Miller et al., 2011). Analysis of

total dietary fibre (AOAC Method 991.43) of the extruded breakfast cereals

showed that RS4 wheat starch retained about 78–89% of its fibre content after

extrusion, with an average fibre retention of around 83%. Product density

increased as the level of RS4 wheat starch increased, but internal cell wall

thickness and size or porosity were not affected. Higher addition of levels of

15% and 20% RS4 wheat starch decreased cereal ring diameter, but increased

initial (dry) cereal crispness and extended bowl life.

While not considered a food-grade starch in the USA and other countries,

starch citrate was evaluated for its performance in pasta and bakery products

by Wepner et al. (1999). Starch citrates from different botanical sources

(potato, pea, wheat, and corn), with 12.2–14.4% esterified citric acid and with

RS content ranging from 45.9–57.5%, were formulated in toast bread by

replacing 10% of the flour in the formula. Toast breads enriched with starch

citrate appeared more yellow in colour, exhibited coarser crumb structure and

possessed slightly lower loaf volume than the control bread. Approximately

60–85% retention of RS content was observed during the preparation of toast

bread.

In another type of baked product, wafers prepared by replacing 7.5–15.0%

of flour with starch citrate displayed a lower percent retention (around 50%) of

RS content compared to toast bread. The sensory attributes of the starch

citrate-fortified wafers were described as harder in texture, lighter in colour,

friable and more brittle, compared to the control wafer.

The addition of 7.5–15.0% starch citrate in a pasta formula caused a

reduction in quality, as demonstrated by reduced firmness of the cooked pasta

and increased cooking loss (Wepner et al., 1999). Twin-screw extrusion of

starch citrates at low shear force resulted in retention of 52–85% of the


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theoretical RS content. By contrast, extrusion at high shear force showed

lower retention (29–65%) of the theoretical RS content. The extrudates in both

treatments had a comparable expansion index, water absorption index and

water solubility index, but extrusion at a low shear force caused a higher

breaking force (13.66–33.28N) compared to extrusion at a high shear force

(7.79–23.40N).

3.7 CONCLUSIONS AND FUTURE PERSPECTIVES

Physical activity, portion control and wholesome foods define a person’s

healthy lifestyle. While the first two items are behavioural in nature, the third

can be designed in foods. With health and wellness soundly resonating with

consumers, food developers are constantly challenged to design consumer

food products that not only have superior taste and texture, but are also

nutritious.

Resistant starch ingredients, in general, provide an easy solution to assist

food scientists in tailoring foods with remarkable end-use qualities and that

address health concerns. Since Hi-MaizeTM was released in 1993 as the first

resistant starch ingredient, the growth of RS has been phenomenal, with at

least 30 RS ingredients now sold and marketed worldwide. As discussed in

this chapter, a range of RS4-type resistant starches, in addition to the other

categories of RS, are available in the tool box of food designers. Prevailing

evidence confirms a host of physiological benefits and food product func-

tionalities of RS4-type resistant starches. There are opportunities for research

scientists and food technologists to identify additional useful attributes of

commercially-available resistant starch ingredients other than for caloric

reduction or as a fibre source.

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4 Novel Applications of Amylose-LipidComplex as Resistant Starch Type 5

Jovin Hasjim1, Yongfeng Ai2 and Jay-lin Jane2

1Queensland Alliance for Agriculture and Food Innovation, Centre forNutrition and Food Sciences, The University of Queensland, Australia

2Department of Food Science and Human Nutrition, Iowa State University,USA

4.1 INTRODUCTION

Amylose is known to form single helical complexes with many other sub-

stances, including iodine, monoglycerides, lysophospholipids, fatty acids and

alcohols. Amylose-lipid complex is commonly found in native starch granules

and processed starch (Becker et al., 2001; Morrison et al., 1993, 1984). The

hydrocarbon chain of the lipid interacts with the hydrophobic moiety of the

amylose chain and fills the central cavity of the amylose single helix (Jane &

Robyt, 1984; Jane et al., 1985; Morrison et al., 1993). The number of glucose

units per helical turn ranges from six to eight, depending on the size of the cross-

section of the complexing agent (Jane & Robyt, 1984; Shogren et al., 2006).

Amylopectin can also form a single helical complex with lipids. Cold-

water soluble starch prepared by heating normal maize starch in aqueous

alcohol solution shows the V-type X-ray diffraction pattern associated with

starch-lipid complex (Jane et al., 1986a, 1986b). The degree of crystallinity of

the cold-water soluble maize starch is higher than its amylose content, which

implies that amylopectin is involved in the complex formation with alcohols.

The presence of amylopectin-lipid complex also restricts the reassociation of

amylopectin branches into double helices, as observed in the decrease of the

degree of starch retrogradation during storage (Eliasson & Ljunger, 1988;

Huang & White, 1993).

Amylose-lipid complex is resistant to amylolytic enzyme hydrolysis

(Holm et al., 1983; Jane & Robyt, 1984; Kitahara et al., 1996; Seneviratne

& Biliaderis, 1991). It has been proposed as a new source of resistant starch

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(RS, type 5) in addition to the four earlier proposed sources (i.e. physically

inaccessible starch (RS type 1), native B- and some C-type polymorphic starch

granules (RS type 2), retrograded amylose (RS type 3) and chemicallymodified

starch (RS type 4)) (Eerlingen & Delcour, 1995; Englyst et al., 1992).

Amylose-lipid complex has been shown to reduce postprandial glycemic

and insulinemic responses (Hasjim et al., 2010; Holm et al., 1983; Takase

et al., 1994). Removal of lipids from native high-amylose maize starch (RS

type 2) also results in a decrease in the enzyme resistance of the starch (Jiang

et al., 2010). The advantages of amylose-lipid complex as RS comparing with

other types of RS are that:

1. amylose-lipid complex is more heat stable than most native B- and C-type

polymorphic starch granules (RS type 2) such as potato and banana starches;

2. the production of amylose-lipid complex requires less extensive physical

and/or chemical processing than that of retrograded amylose (RS type 3)

and chemically modified starch (RS type 4);

3. amylose-lipid complex restores its complex structure spontaneously

during cooling after being heated above its dissociation temperature

(Biliaderis & Galloway, 1989; Hasjim et al., 2010).

4.2 ENZYME DIGESTIBILITY OF AMYLOSE-LIPIDCOMPLEX

Amylose-lipid complex is more resistant to amylolytic enzyme hydrolysis

than amorphous amylose and most A-type polymorphic starches. The enzyme

resistance of amylose-lipid complex depends on the molecular structures of

the complexing lipid and the amylose (Gelders et al., 2005; Kitahara et al.,

1996) and on the crystalline structure of the amylose-lipid complex

(Seneviratne & Biliaderis, 1991). In starch granules, amylose-lipid complex

has been shown to retard granule swelling during heating in excess water,

which reduces the enzyme accessibility to hydrolyze the starch granules

(Cui & Oates, 1999; Tester & Morrison, 1990).

In a recent study, cooking of tapioca, normal maize and high-amylose

maize starches in the presence of corn oil, soy lecithin, palmitic acid (PA),

stearic acid (SA), oleic acid (OA) or linoleic acid (LA) was shown to reduce

the susceptibility of the starch to enzyme hydrolysis (Ai et al., 2013). The

same phenomenon, however, was not observed with waxy maize starch. The

results indicate that the amylose-lipid complex formed during the cooking of

non-waxy starch and lipid was responsible for the decrease in the enzyme

digestibility of starch. The presence of amylose-lipid complex was later

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confirmed from the endothermic peak of the dissociation of amylose-lipid

complex by differential scanning calorimetry (DSC) for starch samples

cooked with soy lecithin, PA, SA, OA, and LA. The amylose-lipid complex

in the cooked starch-corn oil mixture was confirmed by 13C nuclear magnetic

resonance (NMR) spectroscopy.

4.2.1 Effects of lipid structure on the enzyme resistanceof amylose-lipid complex

The properties of amylose-lipid complex, including the dissociation temper-

ature and the susceptibility to enzyme hydrolysis, are affected by the

molecular structure of the complexing lipid. In general, the dissociation

temperature of amylose-lipid complex increases with the hydrocarbon-chain

length of the fatty acid (FA) (Biliaderis & Galloway, 1989; Raphaelides &

Karkalas, 1988; Tufvesson et al., 2003), which reflects the stability of the

amylose-lipid complex, i.e. the hydrophobic interaction between amylose and

the hydrophobic chain of the FA. Short-chain and some medium-chain FAs

(up to 10 carbon units) are not as effective complexing agents as long-chain

FAs (12 carbon units and above). This is due to the greater water solubility of

the short- and medium-chain FAs, allowing the FAs to participate in the

aqueous solution instead of complexing with amylose (Tufvesson et al., 2003;

Yotsawimonwat et al., 2008).

The dissociation temperature of amylose-lipid complex also decreases with

the increase in the number of cis double bonds in the FA chain of complexing

lipid (Karkalas et al., 1995; Raphaelides & Karkalas, 1988; Tufvesson et al.,

2003). The kink structure of cis double bond disrupts the alignment of the FA

in the cavity of amylose single helix. The dissociation temperature of amylose

complexed with an unsaturated FA (e.g. OA and LA) is lower than that of

amylose complexed with a saturated FA of the same hydrocarbon chain-length

(e.g. SA; Tufvesson et al., 2003). Consequently, a saturated long-chain FA

produces a more enzyme-resistant amylose complex than a shorter or an

unsaturated FA (Kitahara et al., 1996).

The low water solubility and the high melting temperature of long-chain

FAs, however, reduce the dispersion of these FAs in water and reduces

their availability in the aqueous solution to form complex with amylose

(Yotsawimonwat et al., 2008). The effectiveness of the complex formation

between amylose and long-chain FA can be improved by increasing the

solubility of the long-chain FA at the pH above the pKa of the FA. Long-

chain FAs are more soluble in aqueous solution when they are in salt form,

carrying a negative charge (Tufvesson et al., 2003; Yotsawimonwat et al.,

2008).

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4.2.2 Effects of crystalline structure on the enzymeresistance of amylose-lipid complex

On the basis of crystallinity, there are two forms of amylose-lipid complex.

The amorphous (Form I) complex is produced by mixing amylose and lipid at

a lower temperature (25–60 �C), whereas the crystalline (Form II) complex

is produced at a higher temperature (90–100 �C) (Gelders et al., 2005;

Seneviratne & Biliaderis, 1991; Tufvesson et al., 2003). Consequently, the

amorphous complex has a lower dissociation temperature (<100 �C) than

does the crystalline counterpart (>100 �C). Amorphous amylose-lipid com-

plex can be converted to crystalline complex through isothermal annealing at a

temperature above the dissociation temperature of the amorphous complex

and below that of the crystalline complex.

The enzyme resistance of amylose-lipid complex can be attributed to

the collapsed helical conformation of the starch-lipid complex, preventing

the dispersion of amylose molecules and interfering with the binding of

amylose molecules by enzyme for hydrolysis (Jane & Robyt, 1984).

In addition, the crystalline structure of amylose helical complex protects

the bulk of the amylose in the crystallites and enhances the enzyme resistance

of the amylose-lipid complex. Hence, the crystalline amylose-lipid complex

(Form II) is more resistant to amylolytic enzyme hydrolysis than the amor-

phous complex counterpart (Form I) (Gelders et al., 2005; Seneviratne &

Biliaderis, 1991).

4.2.3 Effects of amylose-lipid complex on the enzymeresistance of granular starch

Amylose-lipid complex is commonly found in native cereal starch granules

(Morrison et al., 1993, 1984). The native lipids in cereal starch granules,

including maize, rice, barley, oat, millet, and sorghum, are mostly free FAs

and lysophospholipids, and the content of lipids in cereal starch granules is

positively correlated with the amylose content of the starch (Morrison et al.,

1984). These lipids are found on the surface as well as in the inner part of

starch granules (Morrison, 1981). Amylose-lipid complex of granular starch

usually shows a dissociation temperature ranging between 70–108 �C, asanalyzed using DSC in the presence of excess water (Kugimiya et al., 1980; Li

et al., 2008).

The physicochemical properties of native starch granules are greatly

affected by the presence of amylose-lipid complex, including the decreases

in granule swelling, molecular leaching and enzyme digestibility. The forma-

tion of amylose-lipid complex with a helical conformation enhances the

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entanglements between amylose and amylopectin molecules, restricting

amylose leaching and retarding the swelling of starch granules during heating

in excess water (Becker et al., 2001; Cui & Oates, 1999; Tester & Morrison,

1990). Consequently, it reduces the accessibility of enzyme to hydrolyze the

starch (Cui & Oates, 1999). Furthermore, a decrease in RS content was

observed when native high-amylose maize starch was defatted using methanol

solution (Jiang et al., 2010).

4.3 PRODUCTION OF RESISTANT GRANULAR STARCHTHROUGH STARCH-LIPID COMPLEX FORMATION

A technology of producing RS by increasing the amount of amylose-lipid

complex (RS type 5) in granular starch has been recently developed. The

process includes a treatment of swollen starch granules with a debranching

enzyme (isoamylase or pullulanase) to remove the branch linkages of

amylopectin. The resulting free long branch-chains of amylopectin can

function in a similar way to amylose molecules; thus, they can effectively

complex with FA. High-amylose maize starch VII (HA7) was chosen for this

technology because it consists of large proportions of amylose and long

branch-chains of amylopectin (Jane et al., 1999; Li et al., 2008).

A suspension of HA7 is pre-heated at an elevated temperature and is then

incubated with a debranching enzyme – either isoamylase (ISO) or pullula-

nase (PUL). The ISO- or PUL-treated-HA7 suspension is then mixed with FA

at a temperature above the melting temperature of the FAwith stirring for 30

minutes to allow the amylose-lipid complex formation. The complex is

recovered by centrifugation, and the excess FA is removed by washing

with 50% ethanol. FAs are effective complexing agents for making RS

and are economically favourable, because FAs are often the by-products of

edible oil refining process. The resulting HA7-FA complex products have RS

contents up to 75%, measured using the AOAC Method 991.43 for total

dietary fibres, in which the each complex product was hydrolyzed for

30 minutes with a thermally stable a-amylase in a boiling water bath

(Horwithz, 2003; see Table 4.1). The effects of debranching and complex

formation with FA on the RS contents are significant at p< 0.01.

4.3.1 Effects of fatty-acid structure on the RS content

The increase in the RS content of HA7 after complex formation with FA

(Table 4.1) is attributed to the increased amount of amylose-lipid complex (RS

type 5). PA and SA are preferred for the production of RS type 5 because of


CH04GR 07/29/2013 17:14:45 Page 84

their long hydrocarbon chains and straight molecular structures, which

form stable inclusion-complexes with amylose (Tufvesson et al., 2003;

Yotsawimonwat et al., 2008).

When short-chain FAs, such as sodium propionate and butyric acid (BA),

are used to form helical complexes with HA7, the products display lower RS

contents than those made with PA and SA (Table 4.1). The differences in the

RS contents are attributed to the greater water solubility of short-chain FAs,

preventing the formation of stable helical complexes with amylose. As a

result, the order of the RS contents of the complex products is: SA complex>PA complex > myristic acid (or MA) complex > BA complex.

Unsaturated FAwith cis double bond(s) (e.g. OA, which has a kink in the

hydrophobic chain), is less effective for amylose-lipid complex formation

(Karkalas & Raphaelides, 1986; Tufvesson et al., 2003) and produces a

complex product with a lower RS content (63.2%) than the complex product

of saturated FA with the same carbon number (SA, 71.6%) (Table 4.1).

The RS contents of pre-heated HA7 control and (PUL-treated HA7)-FA

complex products are substantially reduced after they are defatted using 85%

methanol solution in a Soxhlet extractor for 24 hours (Table 4.2). These results

support the fact that amylose-lipid complex contributes to the enzyme

Table 4.1 Resistant starch contents of high-amylose maize starch VII after pre-heating at80 �C, treatment with debranching enzyme and complexing with fatty acid at 80 �C.1

Treatments2 Resistant starch content (%)3

Pre-heated HA7 control 36.7�0.2HA7-SA complex 59.8�2.8HA7-PA complex 58.3�1.7ISO-treated HA7 57.8�0.1PUL-treated HA7 46.0�1.6(ISO-treated HA7)-SA complex 74.8�1.5(ISO-treated HA7)-PA complex 74.3�2.4(PUL-treated HA7)-SA complex 71.6�0.3(PUL-treated HA7)-PA complex 69.9�2.2(PUL-treated HA7)-MA complex 62.7�3.0(PUL-treated HA7)-BA complex 44.8�0.8(PUL-treated HA7)-NaPr complex 48.1�2.9(PUL-treated HA7)-OA complex 63.2�0.6

1Values are means� standard deviations.2HA7¼ high-amylose maize starch VII, SA¼ stearic acid, PA¼palmitic acid, ISO¼ isoamylase, PUL¼pullulanase, MA¼myristic acid, BA¼butyric acid, NaPr¼ sodium propionate, and OA¼oleic acid.3Resistant starch content was analyzed following the AOAC Method 991.43 for total dietary fibres

using thermally stable a-amylase in a boiling water bath.

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resistance of the control HA7 starch and the (PUL-treated HA7)-FA complex

products. The RS contents of the defatted (PUL-treated HA7)-FA complex

products are, however, higher than those of the pre-heated HA7 control, with

andwithout defatting, which suggests that the FA in the (PUL-treatedHA7)-FA

complex products cannot be completely removed by the defatting process.

These results further support the complex formation between the PUL-

treated HA7 starch and FA because the FA in starch-lipid complex is less

extractable than uncomplexed FA (Karkalas & Raphaelides, 1986). In addi-

tion, the defatted (PUL-treated HA7)-SA complex product shows a substan-

tially higher RS content than the defatted (PUL-treated HA7)-PA complex

product, which could be attributed to the lower extractability of the longer-

chain FA (SA) in the starch-lipid complex than the shorter one (PA).

4.3.2 Effects of debranching on the RS content

The RS contents of the HA7 starch and the HA7-FA complex products

increase after the HA7 starch is debranched with ISO or PUL (Table 4.1).

Debranching enzyme (ISO or PUL) hydrolyzes the a-(1! 6) glycosidic

linkages and releases linear chains from amylopectin and also from amylose.

The free linear chains have greater mobility to associate between themselves

to form double helices, resulting in the increase of RS content as shown in

the ISO-treated and PUL-treated HA7 starch products (without complexing

with FA).

A combination of the debranching reaction and the complex formation

with FA further increases the RS content of HA7 starch; examples are

Table 4.2 Resistant starch contents of high-amylose maize starch VII-fatty acid complexproducts after defatting.1

Resistant starch content (%)3

Treatments2 Before defatting After defatting Difference

Pre-heated HA7 control 36.7�0.2 30.2�1.3 6.5(PUL-treated HA7)-PA complex 69.9�2.2 38.2�0.7 31.7(PUL-treated HA7)-SA complex 71.6�0.3 50.9�0.4 20.7

1Defatting was carried out using 85%methanol solution in a Soxhlet extractor for 24 hours. Values are

means� standard deviations.2HA7¼ high-amylose maize starch VII, PUL¼pullulanase, PA¼palmitic acid, and SA¼ stearic acid.

HA7 starch was pre-heated at 80 �C before treatments with PUL and fatty acids.3Resistant starch content was analyzed following the AOAC Method 991.43 for total dietary fibres,

using thermally stable a-amylase in a boiling water bath.


CH04GR 07/29/2013 17:14:45 Page 86

(ISO-treated HA7)-FA and (PUL-treated HA7)-FA complex products

(Table 4.1). These results show a synergistic effect of the two treatments,

in which the linear chains produced from the debranching reaction can

form complex more efficiently with FA than can the highly branched

amylopectin molecules.

The complex formation between FAs and relatively short linear dextrins

produced from the debranching of amylopectin in waxy rice starch has been

reported (Yotsawimonwat et al., 2008). Because most high-amylose maize

starches, including HA7 starch, have amylopectin with long branch-chains

(Jane et al., 1999; Li et al., 2008), some linear long chains released from the

debranching of HA7 amylopectin are expected to function in a similar fashion

to amylose. This is supported by the ability of the starch from double-mutant

ae-waxy maize (which consists of only amylopectin, with similar structure to

the HA7 amylopectin) to form complex with iodine and give a blue colour

equivalent to 34.5% apparent amylose content (Jane et al., 1999).

The highly branched structure of amylopectin molecules creates steric

hindrance, which prevents the formation of highly ordered amylopectin-lipid

complex. Thus, the long-branch chains of amylopectin, after being released

from the parent molecules through the debranching reaction, can form helical-

complex with lipids more effectively.

4.4 APPLICATIONS OF THE RS TYPE 5

The (ISO-treated HA7)-FA and (PUL-treated HA7)-FA complex products

contain RS up to 75% after enzyme digestion at 100 �C in excess water

following the AOAC Method 991.43 (Table 4.1). The results indicate that RS

type 5 in the (ISO-treated HA7)-FA and (PUL-treated HA7)-FA complex

products are thermally stable and, thus, are suitable for food processing that

requires heating.

In addition, the heat stability of an (ISO-treated HA7)-PA complex product

has been tested in bread making. In a white bread recipe, 72% (dry basis, db)

of wheat flour was replaced with (ISO-treated HA7)-PA complex product

(60%, db) and wheat gluten (12%, db) (Hasjim et al., 2010). The analytical

result of the RS content of the bread containing RS type 5, determined using

the AOAC Method 991.43, was 34.4% (db), which was similar to the value

calculated (33.6%, db) from the RS contents of the wheat flour, (ISO-treated

HA7)-PA complex product and wheat gluten used to make the bread (Table

4.3). Although bread made with such a high content of RS type 5 was not

intended for a desirable texture, the result proves that RS type 5 is heat stable

and that the enzyme resistance remains in the bread after baking.

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CH04GR 07/29/2013 17:14:46 Page 87

4.5 HEALTH BENEFITS OF RS TYPE 5

The high contents of RS type 5 in the (ISO-treated HA7)-FA and (PUL-treated

HA7)-FA complex products from in vitro studies (Tables 4.1 and 4.2) suggest

that they have potential health benefits in controlling postprandial glycemic

and insulinemic responses and preventing colon cancer (Higgins, 2004;

Nugent, 2005). Human and animal studies have been conducted to investigate

the health benefits of the RS type 5 (Hasjim et al., 2010; Zhao et al., 2011).

4.5.1 Glycemic and insulinemic control

Ahuman feeding study was conducted to understand the effects of RS type 5 on

postprandial glycemic and insulinemic responses (Hasjim et al., 2010). The two

types of breads used in the study were control white bread and RS-type-5 bread

containing 60%, db, (ISO-treated HA7)-PA complex product (Table 4.3). The

results showed that the total areas under the curves of the postprandial glycemic

and insulinemic responses were reduced to 55% and 43%, respectively, when

the human subjects ingested the bread containing RS type 5, compared with the

control white bread (as 100%) (Figure 4.1). Similarly, reduced postprandial

insulinemic response was also observed from the rats fed with starch-mono-

stearoylglycerol complex, which was accompanied by declined lipogenesis in

the adipose tissue and liver (Takase et al., 1994).

The results from the in vivo studies indicate that RS type 5 can be used to

prevent the occurrence of hyperglycaemia and hyperinsulinaemia, which also

Table 4.3 Resistant starch content of breadmade from RS type 5. Reprinted fromHasjimet al., 2010.

Resistant starch (%)

Samples Analytical1 Calculated2

Wheat flour 2.1 –(ISO-treated HA7)-PA complex 52.7 –Wheat gluten 11.0 –Control white bread3 3.3 2.0RS type 5 bread4 34.4 33.6

1Resistant starch analyzed using the AOAC Method 991.43.2Resistant starch calculated on the basis of resistant starch contents of wheat flour, (ISO-treated HA7)-

PA complex product and wheat gluten.3Control white bread contained 96% wheat flour.4Enzyme-resistant bread contained 24% wheat flour, 60% (ISO-treated HA7)-PA complex product and

12% wheat gluten.


CH04GR 07/29/2013 17:14:46 Page 88

prevents the hyperglycaemia-induced hypoglycaemia and the constant feeling

of hunger associated with it (Cryer, 1999; Weinger et al., 1995). Furthermore,

the results showed that the insulinemic responsewas reduced to 2mU/L (close

to the baseline) 120 minutes after ingesting the bread containing RS type 5,

versus about 12mU/L after ingesting the white bread (Figure 4.1; Hasjim

et al., 2010). This suggests that RS type 5 may prevent the development of

insulin resistance, an abnormality caused by persistent hyperinsulinaemia

from frequent consumption of high glycemic-response foods (Byrnes et al.,

(a) Glycemic response

(b) Insulinemic response

RS5 breadWhite bread

RS5 bread

White bread

Time (min)

Incr

ease

in g

luco

se (

mg/

dL)

Incr

ease

in in

sulin

(m

U/L

)

40

35

30

25

20

15

10

5

0

30

25

20

15

10

5

0

–15 0 15 30 45 60 75 90 105 120

Time (min)–15 0 15 30 45 60 75 90 105 120

45

Figure 4.1 (a) Average postprandial glycemic and (b) insulinemic responses of 20male human subjects after ingesting control white bread and bread containing 60%(ISO-treated HA7)-PA complex product (RS type 5). Reprinted from Hasjim et al.,2010.

88 Resistant Starch

CH04GR 07/29/2013 17:14:46 Page 89

1995; Higgins, 2004). Thus, RS type 5 has a potential for interventions of

metabolic syndrome including type 2 diabetes, obesity, hypertension, lipid

abnormalities and heart disease, which have been associated with repeated

hyperinsulinaemia and insulin resistance (Higgins, 2004; Ludwig, 2002;

Takase et al., 1994).

4.5.2 Colon cancer prevention

Potential gut health benefits of RS type 5 were studied using an animal model,

and the (PUL-treated HA7)-SA complex product was used as the source of RS

type 5 (Zhao et al., 2011). Normal maize starch (NMS) and HA7 were used as

negative and positive controls, respectively. The starches were heated in

excess water (1 g starch in 3 g water) on a stove until they were boiling or

formed consistent starch paste. After cooking, the starches, at 55% (db) of the

total weight, were mixed with other ingredients to prepare NMS, HA7 and RS-

type-5 diets following the recipe of AIN-93 Rodent Diets (Reeves, 1997)

without the addition of cellulose.

Three groups of five-week-old male Fischer-344 rats were injected with

two doses (20mg/kg body weight) of azoxymethane (AOM) in oneweek apart

and fed with NMS diet until three days after the second injection, followed by

a treatment diet (NMS, HA7, or RS-type-5 diet). After eight weeks of feeding

on the treatment diet, the rats were sacrificed, and the occurrence of mucin-

depleted foci (MDF) and aberrant crypt foci (ACF) in the colon as well as the

caecum digesta weight and pH were analyzed. MDF and ACF are commonly

considered as the precursor lesions of chemically induced colon cancer

(Arakaki et al., 2006; McLellan et al., 1991).

It was found that the average number of MDF in the colons of the rats fed

with the RS-type-5 diet was significantly lower than those in the colons of the

rats fed with the NMS and HA7 diets (Table 4.4). The average number of ACF

was also reduced by the RS-type-5 diet, although the number was not

significantly different from those of the other diets. A significantly larger

amount of caecal digesta and a lower caecum pH were observed in the group

fed with RS type 5, compared with those in the other two groups (Table 4.4).

Lowering the pH of digesta may create an environment that suppresses the

growth of pathogenic organisms in the colon (Kleessen et al., 1997; Silvi

et al., 1999). No significant differences were found in the average daily food

intake and body weight gain among the rats fed with different diets in this

study.

In another study using the same animal model and diets, it was found that a

significantly larger amount of faeces was discharged by the rats fed with RS

type 5 than by those fed with the other two diets (Table 4.5). In addition, the


CH04GR 07/29/2013 17:14:46 Page 90

faeces from the rats fed with RS type 5 contained significantly greater

amounts of starch and lipid than those from the other two groups fed with

NMS and HA7. Polarized-light micrographs of the faeces collected from the

rats fed with RS type 5 showed starch granules with birefringence (Figure

4.2). The results of gas chromatography showed that SA was the major

component of the lipids extracted from the faeces of the rats fed with RS type

5, indicating that a proportion of the (PUL-treated HA7)-SA complex product

was not digested in the small intestine and was not fermented in the colon

(Table 4.5). Therefore, SA was not absorbed in the digestive tract and was

discharged as starch-lipid complex. The results also suggest that 15% (db) of

Table 4.4 Mucin-depleted foci, aberrant crypt foci and caecum digesta weight and pHfrom rats injected with azoxymethane as colon cancer inducer (Zhao et al., 2011).1

Adapted from Zhao et al., 2011. Copyright 2011 American Chemical Society.

Diet2

Mucin-depleted foci(MDF)

Aberrantcrypt foci(ACF)

Caecaldigestaweight (g)

CaecumdigestapH

Cooked normalmaize starch

3.5�1.8 47.5�13.4 3.29 7.5�0.1

Cooked HA7 1.8�1.4� 39.6�18.0 13.59� 6.6�0.6�

Cooked (PUL-treatedHA7)-SA complex

0.3�0.5� 30.9�25.0 18.67� 5.7�0.2�

1Values are means� standard deviations.2Each diet contained 55% (db) starch.�Significant at p<0.05 compared with cooked normal maize starch diet.

Table 4.5 Daily weight, total starch content, and lipid content of faeces collected fromrats fed with different diets.1

Faeces weight (g/day) Starch contentof faeces (%)3

Lipid contentof faeces (%)4

Diet2 As is Dry

Cooked normalmaize starch

0.23�0.07 a 0.20�0.06 a 0.2�0.0 a 16.8�0.4 ab

Cooked HA7 0.55�0.31 a 0.45�0.25 a 16.8�1.0 b 11.7�0.3 aCooked(PUL-treated

HA7)-SAcomplex2.04�0.76 b 1.65�0.62 b 44.0�0.0 c 22.0�2.2 b

1Values are means� standard deviations. Different letters on the same column represent significant

difference at p<0.05.2Each diet contained 55% (db) starch.3Starch contentwasanalyzedusingMegazymeTotal StarchAssayKit following theAACCMethod76-13.4Lipid content analyzed using the AOAC Method 996.06.

90 Resistant Starch

CH04GR 07/29/2013 17:14:46 Page 91

the consumed RS type 5 was not utilized by the rats and the microbes residing

in the digestive tract of the rats. The inferior digestibility of the RS type 5,

however, did not affect the body weight gain of the rats.The results from these two in vivo rat studies suggest two possible

mechanisms of RS type 5 in reducing the amount of MDF in the colon.

One is the greater amount of the faeces discharged from the colon. The

larger, bulky amount of faeces can purge carcinogen (e.g. the injected

AOM) out from the colon. The other is the FA present in the starch-lipid

complex, which may have the affinity to absorb carcinogen and to remove

it from the colon.

4.6 CONCLUSION

Amylose-lipid complex has been proposed as RS type 5, because of its

resistance to enzyme hydrolysis. The presence of amylose-lipid complex in

starch granules also increases their enzyme resistance by restricting the

granule swelling during cooking. The enzyme resistance of amylose-lipid

complex depends on the molecular structure of the lipid and the crystalline

structure of the single helices. FA with a longer hydrocarbon chain length

produces amylose-lipid complex with greater enzyme resistance, whereas FA

with a greater degree of unsaturation produces amylose-lipid complex with

Figure 4.2 Polarized-light micrograph of starch isolated from faeces of rats fed with(PUL-treated HA7)-SA complex product.


CH04GR 07/29/2013 17:14:47 Page 92

lower enzyme resistance. Furthermore, a crystalline form of amylose-lipid

complex has greater enzyme resistance than its amorphous counterpart.

A technology has been recently developed to produce RS using amylose-

lipid complex by treating granular HA7 starch with debranching enzyme,

followed by complex formation with FA. The RS contents of the complex

products were significantly higher (up to 75%). Feeding human subjects with

this RS type 5 reduced postprandial glycemic and insulinemic responses

compared with the control diet of white bread, which suggests that RS type 5

has potential to intervene in metabolic syndromes including type-2 diabetes,

obesity, hypertension, lipid abnormalities and heart disease, as well as other

complications associated with repeated hyperinsulinaemia and insulin resist-

ance. RS type 5 also shows the ability to reduce colon cancer development, as

observed by lowering the amount of MDF in the colons of AOM-treated rats.

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Biliaderis, C.G., Galloway, G. (1989). Crystallization behavior of amylose-V complexes:Structure-property relationships. Carbohydrate Research 189, 31–48.

Byrnes, S.E., Brand Miller, J.C., Denyer, G.S. (1995). Amylopectin starch promotes thedevelopment of insulin resistance in rats. Journal of Nutrition 125(6), 1430–1437.

Cryer, P.E. (1999). Symptoms of hypoglycemia, thresholds for their occurrence, andhypoglycemia unawareness. Endocrinology & Metabolism Clinics of North America28(3), 495–500.

Cui, R., Oates, C.G. (1999). The effect of amylose-lipid complex formation on enzymesusceptibility of sago starch. Food Chemistry 65(4), 417–425.

Eerlingen, R.C., Delcour, J.A. (1995). Formation, analysis, structure and properties of typeIII enzyme resistant starch. Journal of Cereal Science 22(2), 129–138.

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Hasjim, J., Lee, S.-O., Hendrich, S., Setiawan, S., Ai, Y., Jane, J. (2010). Characterizationof a novel resistant-starch and its effects on postprandial plasma-glucose and insulinresponses. Cereal Chemistry 87(4), 257–262.

Higgins, J.A. (2004). Resistant starch: Metabolic effects and potential health benefits.Journal of AOAC International 87(3), 761–768.

Holm, J., Bj€orck, I., Ostrowska, S., Eliasson, A.C., Asp, N.G., Larsson, K., Lundquist, I.(1983). Digestibility of amylose-lipid complexes in-vitro and in-vivo. Starch - St€arke35(9), 294–297.

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Jane, J.-L., Craig, S.A.S., Seib, P.A., Carl Hoseney, R. (1986a). A granular coldwater-solublestarch gives a V-type X-ray diffraction pattern. Carbohydrate Research 150(1), C5–C6.

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5 Digestion Resistant Carbohydrates

Annette EvansInnovation and Commercial Development, Tate & Lyle, USA

5.1 INTRODUCTION

The rates of obesity, diabetes, cardiovascular and other diseases have

increased dramatically over the last century, which has led to increased

consumer awareness of the importance of nutrition in the development and

progression of these diseases. As a result of this awareness, and increased

scientific research in this area, demand for ‘healthier options’ is high.

Consumers understand the value and potential health benefits of dietary fiber,

so one top demand is for fiber-containing foods that taste good. For food

ingredient companies, this presents both an opportunity and a challenge to

supply the market with new and nutritionally improved carbohydrate ingre-

dients, including starch and fiber.

To develop fiber-containing ingredients, it is important to understand the

enzymes involved in food digestion, as well as the properties of a food that

make it either highly digestible or highly enzyme-resistant and, therefore, high

in dietary fiber. In the case of starch, the structures responsible for enzyme

resistance can be either physical or molecular. This chapter examines the

physical and molecular structures of starch that influence enzyme digestibil-

ity, and describes examples of commercially available resistant starch ingre-

dients and their source of enzyme resistance related to starch structure.

5.2 STARCH DIGESTION

Starch is the major source of carbohydrates in the human diet. Starch can be

obtained from fruit, vegetables, roots and grains. Starch and starch derivatives

are a nutritive, abundant and economical food source. Today, starch is

consumed unprocessed in the form of raw fruits and vegetables, or in the

95




CH05GR 07/29/2013 16:2:52 Page 96

form of more shelf-stable processed foods (Moore et al., 1984). The process-

ing of starch evolved in the last 80 years from a simple isolation process to the

production of diversified lines of sweeteners and starch products (O’Dell,

1979). Food starches contribute to the characteristic viscosity, texture, mouth-

feel and consistency of many food products.

Starch is a glucose polymer composed of highly branched amylopectin

(a-1,4 and a-1,6-linkages) and essentially linear amylose molecules (mainly

a-1,4-linkages) (Figure 5.1).

Starch digestion in the human body is mainly done by a-amylases. Salivary

a-amylase is the first amylase that comes in contact with the food in the

mouth. It catalyzes the hydrolysis of amylose to maltose, maltotriose and

maltotetraose, and the hydrolysis of amylopectin to the same products plus

two a-limit dextrins (Robyt, 1984). The salivary a-amylase quickly passes

from the mouth to the stomach, together with the food starch. The pH in the

stomach is about 2, which inactivates the salivary a-amylase. After some

resident time in the stomach, the partially hydrolyzed starch passes into the

small intestine, where it is neutralized. The main hydrolysis of the starch is

then accomplished by pancreatic a-amylase, secreted from the pancreatic

duct, via a multiple attack mechanism (Mazur & Nakatani, 1993).

Two other enzymes are necessary to convert the hydrolysis products of the

a-amylases into glucose, which can be actively transported across the small

intestine membrane. These two enzymes are the brush border glucogenic

enzymes maltase-glucoamylase and sucrase-isomaltase (Swallow et al., 2001;

Nichols et al., 2003). Maltase-glucoamylase converts maltose, maltotriose,

and maltotetraose into D-glucose by successive action from the non-reducing

O

OH

OH

HHO

HO

H

H H

H

O

O

OH

HHO

H

H H

H

O

O

OH

OHH

OHH

H H

H

O

HOO

OH

OHH

HOH

H H

H

OH

α-1,6-linkage

α-1,4-linkage

Figure 5.1 Structures found in starch molecules.

96 Resistant Starch

CH05GR 07/29/2013 16:2:52 Page 97

end (Robyt, 1984). Sucrase-isomaltase hydrolyses the a-1,6-linkages. Theresulting D-glucose can then actively be transported across the luminal

membrane of the small intestine and thence into the blood via the sodium-

glucose cotransporter (SGLT-1), which is located at the luminal surface of

enterocytes (Pencek et al., 2002).

Starch digestion is influenced by factors that affect enzyme activity and the

susceptibility of the starch substrate to the hydrolytic enzymes (Tester et al.,

2004). Enzyme activity may depend on quantity of the enzymes present,

enzyme isoform and enzyme inhibitors either present in the consumed food or

produced during enzyme hydrolysis of the food. While brush border enzymes

are capable of cleaving both a-1,4 and a-1,6 glycosidic bonds as well as otherglycosidic bonds (e.g. a 1,2), the main hydrolysis of starch is performed by the

a-amylases. Starch digestion by a-amylases requires a series of steps where

the enzymes first need to diffuse to the starch matrix into the food, then bind

to the substrate, then finally cleave the a-1,4-glycosidic linkages (Brayer

et al., 2000).

5.3 PHYSICAL STRUCTURES OF STARCH

To understand factors that may influence the susceptibility of a particular

starch substrate to a-amylase hydrolysis, four aspects of hydrolysis should be

considered: the diffusion of the enzyme towards the substrate; the porosity of

the starch substrate; the adsorption of the enzyme on the substrate; and the

catalytic event (Colonna et al., 1992). Porosity has to be considered at two

levels of scale: at the macro (1–100mm) and at the micro molecular level

(<10 nm) (Colonna et al., 1992). The physical form of the starch also

influences enzyme binding. Physical entrapment of starch in the food matrix,

starch granular structure, and also smaller scale structures such as crystallinity

and helicity, may influence the binding of enzymes to the starch substrate.

For native starch granules, the limiting factor for the hydrolysis has been

shown to be the penetration of the enzyme into the granules by successive

formation of pits and larger pores (Gallant et al., 1973).

Before a substrate can be hydrolyzed, adsorption of the enzyme onto the

substrate is crucial. Any factors that influence the binding of the a-amylases to

the starch substrate can slow down or limit starch digestion. The hydrolysis

products maltose and maltotriose have been shown to act as substrate

inhibitors for a-amylase (Seigner et al., 1985).

For an enzyme to be able to hydrolyze a substrate, proper enzyme-substrate

recognition is necessary. Substrate binding by means of the enzyme sub-sites

has to be energetically favoured. For the catalytic action of the enzyme to

Digestion Resistant Carbohydrates 97

CH05GR 07/29/2013 16:2:52 Page 98

occur, the substrate to be hydrolyzed has to be oriented properly inside the

active side of the enzyme. Only the part of the starch substrate that can fit into

the active site cavity of the a-amylase can be hydrolyzed. Structures such as

double helices are too big to fit into the active site cavity and can only be

hydrolyzed in the unwound state.

5.3.1 Starch helices

Amylose is known to form single helices in solution. The addition of

complexing ligands such as iodine, fatty acids, alcohols, emulsifiers or

flavour compounds to aqueous starch solutions induces the formation of

single helices (Heinemann et al., 2003). V-amylose is the generic term for

amylose obtained by complexation, and the compounds used to obtain the

single helices may or may not be present in the final V-type starch (Buleon

et al., 1998). The amylose single helix has been described as a left-handed

helix with six glucose residues per turn, with a pitch height of about 0.7 nm

(Buleon et al., 1998). The outer surface of the single helix is thought to be

mostly hydrophilic, while the centre channel of the helix is hydrophobic.

Single helices are stabilized by 2-O . . . O-6 hydrogen bonds between

spatially close glucose residues of the next spiral turn (intrahelical hydrogen

bonds). The structure of the single helices is further cooperatively strength-

ened by 2-O . . . O-3 hydrogen bonds between adjacent glucose units

(Immel & Lichtenthaler, 2000).

The formation of singles helices from branched material has been little

studied. Formation of single helices from amylopectin with complexing

agents has been shown (Heinemann et al., 2003). Considering the structure

of amylopectin molecules, it would be expected that external chains that are in

close proximity to each other may easily form double helices.

The external chains of amylopectin and amylose chains are known to be

able to form double helices within starch granules and in non-granular form

(Wu & Sarko, 1978a, 1978b, 1978c). Evidence and models for both right-

handed (Mueller et al., 1995) and left-handed double helices (Imberty et al.,

1988) have been provided. The left-handed form is the energetically favoured

form and is commonly accepted as the form of starch double helices (Imberty

et al., 1988; Buleon et al., 1998). The starch double helix has a pitch of

2.13 nm with six glucose units per turn (Imberty et al., 1988, Buleon et al.,

1998). Starch double helices are stabilized by hydrogen bonds between

2-O . . . O-6 of glucose units on different strands of the double helix

(interhelical hydrogen bonds). The outer surface of the double helix has an

irregular distribution of hydrophobic and hydrophilic areas (Immel and

Lichtenthaler, 2000).

98 Resistant Starch

CH05GR 07/29/2013 16:2:52 Page 99

5.3.2 Crystalline structures

Single helices formed by non-granular starch can arrange themselves to form

crystalline entities. If the crystalline arrays are large enough to diffract x-rays,

an x-ray diffraction pattern can be observed. The characteristic structure

of crystalline V-amylose has been extensively studied (Rappenecker &

Zugenmaier, 2001; Godet et al., 1995). The double helices formed by external

chains of amylopectin or amylose molecules can also form crystalline entities.

Double helical starch can form different crystalline polymorphs. Native

starch granules can be found with A-type crystallinity (cereal starches) and

B-type crystallinity (tubers, high-amylose maize starches) (Banks & Green-

wood, 1975; Zobel, 1988). The double helical structures within the two

polymorphic forms are essentially identical (Imberty et al., 1991), but the

packing of the helices within the crystalline structure is different. In general,

A-type crystals are less hydrated and more densely packed than B-type

crystals (Imberty et al., 1987; Imberty & Perez, 1988).

Once starch has lost its granular structure, it can be crystallized into one of

the two crystalline polymorphs, depending on the starch structure and

crystallization conditions. The A-type polymorph is the more thermo-

dynamically stable form and is preferably formed by shorter chains, higher

temperature and lower starch concentrations. The B-type polymorph is

formed by longer starch chains, lower temperatures and higher starch con-

centrations (Gidley & Bulpin, 1987). B-type crystallites are commonly

believed to be more enzyme resistant, while A-type polymorphs are more

heat stable compared to B-type formed from chains with similar molecular

weight (Whittam et al., 1990; Le Bail et al., 1995).

5.3.3 Starch granule structure

Starch granules are the naturally occurring storage form of starch. Different

levels of organization exist in native starch granules. At the lowest such level,

the starch granules consist of amorphous and semi-crystalline shells that are

often called growth rings. At a higher level of structure are the so-called

‘blocklets’ (Gallant et al., 1997). One blocklet is thought to contain several

amorphous and crystalline lamellae that are, in turn, created by the chain

organization of amylopectin molecules. The side chains of amylopectin

can form double helices and, due to the close proximity of the chains to

each other, the double helices can easily form crystalline arrays. These

crystalline arrays, and the non-crystalline zones between them that have

been shown to contain the amylopectin branch points (Jenkins et al., 1993),

are organized into lamellae.


CH05GR 07/29/2013 16:2:52 Page 100

Heating of starch granules can lead to partial or complete melting of the

crystallites and helices present in the granules. Depending on the heat

treatment conditions used, the heat treatment can be classified as gelatiniza-

tion, annealing or heat-moisture treatment. Gelatinization of starch is defined

as the complete melting of granular structures in the presence of excess water.

Annealing and heat-moisture treatment are both heat treatments that only

partially melt structures in the starch granules by using temperature/moisture

combinations below the melting point of the treated starch.

Annealing is a term used in polymer chemistry to describe a treatment that

uses heat and moisture to increase the order of polymer chains. In the case of

starch, the term ‘annealing’ is used to describe a heat treatment that employs

temperatures below the melting point of the starch at moisture contents above

40%. Heat-moisture treatment of starch is described as a heat treatment with

temperatures below the melting point of starch, at moisture levels below 35%

(Stute, 1992; Jacobs & Delcour, 1998). These treatments are generally used to

‘perfect’ crystalline order in starch granules.

5.4 RESISTANT STARCH DUE TO PHYSICAL STRUCTURE

Resistant starch has been defined as ‘the starch and products of starch

digestion that are not absorbed in the small intestine of healthy individuals’

(Asp, 1992). Based on the source of the enzyme resistance, resistant starch has

been classified into five different types (Table 5.1). In type 2, type 3 and type 5

resistant starches, the enzyme resistance is due to the physical structure of the

starch molecules.

The physical structures responsible for formation of type 2 resistant starch

are part of the natural organization of starch granules. Examples of starches

high in type 2 resistant starch are green banana, potato and high-amylose

Table 5.1 Classification of resistant starch (RS).

RS type Source of enzyme resistance Examples

1 Whole grains Seeds, grains2 Raw starch granule structure Green banana, high-amylose,

potato3 Structures formed on re-association or

retrogradation of starch after heattreatment

Cooked pasta, cooked potato

4 Chemical modifications of the starch Modified starch5 Starch lipid complex Amylose containing starch

100 Resistant Starch

CH05GR 07/29/2013 16:2:52 Page 101

maize. Several commercial type 2 resistant starch ingredients are available

These are usually high-amylose starches that might undergo heat-moisture

treatment to enhance their inherent RS content (US Patent 5593503).

Type 3 resistant starch is formed by reassociation of starch chains after heat

treatment. Different factors, such as amylose/amylopectin ratio, chain length,

lipid content and processing conditions, influence the amount and quality of

the type 3 RS (Eerlingen & Delcour, 1995). Annealing, heat moisture

treatment and gelatinization of starch all melt crystalline structures present

in native starch granules either fully or partially. Upon cooling, linear amylose

molecules, as well as linear regions of amylopectin molecules, organize into a

mix of amorphous areas as well as helices and crystallites, with varying

degrees of enzyme resistance and, therefore, varying levels of resistant starch.

In the case of gelatinization, this reassociation of starch chains upon cooling is

called retrogradation.

The resulting resistant starch often exhibits B-type crystallinity, but A-type

crystalline RS can also be obtained if the retrogradation or crystallization

occurs at high temperatures or involved starch molecules with short chain

length. Formation of crystalline structures that can form RS 3 take place above

the glass transition temperature (and below the melting temperature), and any

components present that influence the glass transition temperature can,

therefore, be expected to influence the formation (yield and quality) of the

formed type 3 RS.

Amylose content has been positively correlated with RS yield (Sievert &

Pomeranz, 1990) and the formation of type 3 RS is strongly related to the

crystallization process of amylose (Eerlingen & Delcour, 1995). The yield of

RS formed is dependent on the water content and temperature used. At high

starch concentrations, the starch chains interact more easily, leading to

increased crystal and RS formation. Water does act as a plasticizer in the

system, and a minimum water content is necessary to achieve the chain

mobility needed to form crystalline structure resistant to enzyme digestion.

The presence of lipids has been shown to decrease the formation of type 3 RS,

due to formation of amylose lipid complexes (type 5 RS) (Czuchajowaska

et al., 1991).

Annealing and heat-moisture treatment can be used for the manufacture

of resistant starch, since the more ‘perfect’ structures formed often lead to

an increase in enzyme resistance of the starch (Jacobs & Delcour, 1998;

Chiu et al., 1999; Brumovsky & Thompson, 2002).

Some commercial examples of type 3 resistant starch are Hi-maize 330

(National Starch and Chem. Co) and Promitor Resistant Starch 60 (Tate &

Lyle). Both of these ingredients are manufactured from high-amylose maize

starch. In both cases, the starch is first gelatinized and then cooled to


CH05GR 07/29/2013 16:2:52 Page 102

retrograde the starch chains. Heat-moisture treatments are then applied to the

retrograded starch to increase the molecular order and, therefore, the enzyme

resistance of the starch (US5281276; US7189288). Type 3 resistant starches

have also been produced by a process in which the starch is first treated with a

debranching enzyme to increase the amount of linear chains, followed by

controlled reassociation or crystallization of the linear chains to form enzyme

resistant starch. One commercially available example of type 3 resistant starch

produced in this fashion is ActistarTM RM (Cargill Inc.), which is produced

by debranching and crystallization of tapioca maltodextrin (Patent EP

0846704 B1).

Tate & Lyle recently described a resistant starch process in which a starch is

first treated with a glucanotransferase enzyme to elongate the external chains

of amylopectin, followed by debranching and then crystallization of the linear

chains (US Patent Application 2007/0059432 A1).

The enzyme resistance in Type 5 resistant starch is due to the molecular

structure of amylose lipid complexes that can either be present in the native

starch or formed by controlled reactions using non-granular starch and

lipids to form the resistant amylose lipid complex (Brown et al., 2006;

Hasjim et al., 2010).

5.5 MOLECULAR STRUCTURE OF STARCH

Starch is comprised of two polymers. Amylose is an essentially linear

polymer, in which glucose monomers are linked by a-1,4-glycosidic bonds.Amylopectin is a highly branched polymer that, in addition to the linear

a-1,4-linkages, also contains a-1,6-linked glucose monomers that create

branch points.

Certain processing treatments alter the molecular structure of starch and

may create additional linkage types. The amount and distribution of a-1,6-branch points can be altered by enzymatic treatment of starch. Debranching

enzymes such as isoamylase or pullulunase can be used to decrease the branch

points, and branching enzymes can be used to increase the amount of a-1,6-linkages in starch. Transferase enzymes can alter the distribution of branch

points in starch.

In addition to altering the amount and distribution of linkages present in

native starch, processing treatments can be used to create new types of

linkages not found in native starch molecules. When heat is applied to starch

at low moisture and low pH conditions, dextrinization of the starch occurs

(Wurzburg, 1986). During the initial stages of dextrinization, acid-catalyzed

hydrolysis occurs. This is followed by a recombination of the fragments to


CH05GR 07/29/2013 16:2:52 Page 103

form branched structures. The dextrinization process converts a portion of the

normal a-1,4-, a-1,6-linkages to random a-1,4-, a-1,6-, a-1,2-, a-1,3 and

even b-type linkages (Wurzburg, 1986). Figure 5.2 shows a representative

molecular structure of a dextrin.

Chemical modification of starch is a process widely used in the food

industry to alter the texture and processing stability of starch. Awide range of

properties can be achieved, depending on the botanical origin of the starch,

amylose and amylopectin structure, and the amount and type of modification

agent. Starch modification can be achieved through derivatization such as

etherification, esterification and cross-linking of starch (Figure 5.3). Chemical

modification involves the introduction of functional groups into the starch

molecules, resulting in significantly different properties. Starches with altered

gelatinization, pasting and retrogradation behaviour can be produced through

chemical modification (Singh et al., 2007).

5.6 ENZYME RESISTANCE DUE TOMOLECULARSTRUCTURE

The linear regions of the starch molecules are easily digested by human

pancreatic a-amylase. The small sugar and oligosaccharide digestion

products of a-amylase can then be digested further by the intestinal brush

border enzymes.

Branch points can create a steric hindrance for enzyme digestion, irre-

spective of the type of glycosidic linkage that creates the branching of the

starch chain. For a-amylase hydrolysis to occur, a linear portion of the starch

chain has to fit in the enzyme’s active site. This linear portion has to be long

enough to create energetically favourable binding. Glucose units close to the

branch points have less favourable binding energy with the sub-sites of the

enzyme than glucose units further down the chain; sub-site binding therefore

affects the efficiency with which the enzyme hydrolyses a linkage close to a

branch point. Linkages directly adjacent may not be hydrolyzed at all, leading

to the formation of limit dextrins (Shannon et al., 1984; Colonna et al., 1992).

Chemical modification of starch can be viewed as a modification that

creates chain irregularities or branches in the starch chains. Cross-linking of

starch covalently links two starch chains together, in effect creating a branch

point on both chains. Chemical substitution introduces a bulky side group to

the starch chains. The introduction of these chemical groups may create a

steric hindrance to one or more human digestive enzymes.

Several type 4 resistant starch ingredients are available commercially, with

total dietary fiber values of up to 90%. These type 4 products, from different


CH05GR 07/29/2013 16:2:53 Page 105

botanical origins, contain high amounts of resistant starch due to a high level

of chemical cross-linking (US 2006/0188631 A1). Substituted starches are not

currently produced as resistant starch ingredients.

Dextrinization of starch leads to the formation of potentially indigestible

linkages. The glycosidic linkages in dextrins are usually predominately a-1,4-, but a-1,6-, a-1,2-, a-1,3 and, possibly, even b-type linkages can be found(Wurzburg, 1986). The a-1,4- and a-1,6-bonds are digestible by human

pancreatic a-amylase and brushborder enzymes, respectively. However,

while some of the a-1,2-bonds might also be digestible by brushborder

enzymes, other bonds formed during dextrinization are not digestible by

human enzymes.

Commercially, the technique of dextrinization is used by several compa-

nies to create dietary fiber ingredients that (depending on their resulting

molecular size) can be classified as resistant starch or resistant maltodextrins.

The Nutriose1 line of dietary fiber ingredients is marketed by Roquette

(Roquette Fr�eres). Nutriose1 soluble fibres are food dextrins made from

wheat or maize starch (US5620871). Fibersol 2 is produced and marketed by

a joint venture between ADM (Archer Daniels Midland Company) and

Matsutani (Matsutani LLC). It is classified as resistant maltodextrin in the

USA (US5358729) and is produced by dextrinization of starch followed by a

proprietary enzymatic molecular weight reduction of the dextrins. Tate &

Lyle’s PromitorTM Soluble Corn Fiber ingredients are produced by heat

treatment of starch hydrolysis products at low pH (US7608436). They can

be classified as corn syrup or maltodextrin, depending on the molecular

weight of the resulting product.

O

HO

OO

O

HO

OO

O

HO

O

O

O

O

OP O

–O

bridgingphosphatediester

O

HO

O

OO

HO

OOOH

bulkyhydroxy-propylether

Figure 5.3 Structures found in modified starch. Left: structure of chemical cross-link(cross-linking with POCl3). Right: structure of chemical substitution (substitution withpropylene oxide).


CH05GR 07/29/2013 16:2:53 Page 106

5.7 CONCLUSION

Carbohydrates play an important role in the human diet for a number of

reasons. The World Health Organization recommends that at least 55% of the

energy of a diet be derived from carbohydrates. Starch is the major source of

carbohydrates in the human diet. Many carbohydrate-containing foods also

contain important micronutrients and phytochemicals.

Dietary fiber is a class of carbohydrates that are important in overall health.

Today, food ingredient companies have the knowledge and expertise to

maximize the health-giving potential of starch ingredients by modulating

not only the texture and sensory properties of commercial starches, but also

the digestibility and physiological effect of a starch ingredient.

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3GCH06 07/20/2013 8:2:37 Page 111

6 Slowly Digestible Starch and HealthBenefits

Genyi Zhang1and Bruce R. Hamaker2

1 School of Food Science and Technology, Jiangnan University, China2Whistler Center for Carbohydrate Research and Department of FoodScience, Purdue University, USA

6.1 INTRODUCTION

Starch is the major glycaemic carbohydrate of foods, and its nutritional

property is related to its rate and extent of digestion and glucose absorption

in the small intestine. A classification of starch into rapidly digestible starch

(RDS), slowly digestible starch (SDS) and resistant starch (RS) has been used

to specify the nutritional quality of starch. SDS is the most elusive fraction of

the three, perhaps due to its somewhat transient nature, and it is certainly less

studied than RS. Yet, the physiological consequences of SDS may be just as

profound as those of RS, in terms of regulating glucose delivery to the body

and its associated metabolic effects, triggering gut hormones to affect

gastrointestinal tract motility and, perhaps, even reducing appetite and

increasing satiety. This chapter covers the current understanding of SDS,

including its concept and potential health benefits, the starch digestion

process, structure and mechanism of SDS and approaches to making SDS.

On one level, available carbohydrates are an important part of the diet and

should provide 45–65% of total caloric intake as recommended in the Dietary

Guidelines for Americans (U.S. Department of Agriculture, 2010). However,

quantity alone is not an accurate assessment of nutritive quality and rate – the

extent of digestion and absorption of glycaemic carbohydrates is an important

parameter (Jenkins et al., 1981; Englyst et al., 2003).

Over thirty years ago, Jenkins et al. (1981) proposed the concept of

glycaemic index (GI), which is represented by area under the in vivo

glycaemic response curve (AUC) of a tested food compared to a reference

glucose or white bread containing the same amount (50 g) of available

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carbohydrate. Glycaemic response is a broader concept that includes the

nature and shape of the postprandial blood glucose profile. The digestion

property of glycaemic carbohydrates is the main determinant of the GI value

of different foods, although factors that affect glucose transport through the

gut enterocytes, as well as other human factors, also impact rate of rise of

blood glucose levels (Wolever et al., 2006).

Brand et al. (1991) categorized foods based on their GI values as high GI

(GI> 70), low GI (GI< 55) and intermediate GI foods. Since the advent of the

GI concept, there have been extensive clinical and nutritional investigations

on dietary carbohydrates, particularly regarding potential health benefits of

low-GI foods (Wolever, 2003), and an extensive list of different foods have

been tested and compiled by Foster-Powell et al. (2002).

There exists a controversy that has developed within both scientific and

industrial groups regarding the use of the GI concept in the context of a tool to

affect public health. This is in large part due to a body of studies with varying

results on the relationship between low-GI food and health (Howlett &

Ashwell, 2008), even though this includes carefully conducted studies with

mechanistic underpinnings. Slowly digestible glycaemic carbohydrates is one

way to achieve low glycaemic response in foods, but it is not the only way. A

better understanding of the structural and mechanistic basis of low glycaemic

response foods will provide important information to this field of study and to

the development of low glycaemic response products.

6.2 SDS AND POTENTIAL BENEFICIALHEALTH EFFECTS

Slowly digestible starch offers the possibility of moderated glucose delivery to

the body, which can be beneficial to glucose homeostasis and related

processes, as well as distal small intestinal glucose release that may affect

motility of food through the gastrointestinal tract and, perhaps, fullness and

satiety. Due to its importance as the central metabolic fuel, levels of glucose

are tightly controlled in the body through a series of mechanisms. Although

glucose may be supplied internally through glycogenolysis and gluco-

neogenesis, dietary derived glucose is critical for growth and development

and for energy-consuming organs such as the brain and central nervous system

(Peters et al., 2004).

Glucose is also a signal molecule in energy metabolism that stimulates

insulin secretion, governs glucose utilization and inhibits endogenous

glucose production. Starch, generally being the most abundant glycaemic


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carbohydrate consumed, is themajor source of dietary glucose. Importantly, the

manner of digestion of starch and subsequent release of glucose in the digestive

system is the starting-point of the glucose homeostasis regulatory cascade, and

its timing and location of release controls glucose delivery of foods.

6.2.1 Potential health benefit of SDS relative to RDS

The nutritional property of starch has been ascribed to relative amounts of RDS,

SDSandRS.RDS results in a largefluctuation inbloodglucose that can generate

stress on the regulatory system of glucose homeostasis (Ludwig, 2002), and this

may even be associated with cell, tissue and organ damages under long-term

stress (Brownlee, 2001). In commonly consumed starchy foods that are proc-

essed, RDS is usually dominant. High RDS correlates with high value of GI and

glycaemic load (GL, GI� amount). Moreover, the high postprandial glycaemic

response elicited by RDS coincides with high levels of plasma insulin, the

principal hormone to regulate postprandial blood glucose levels.

RS, that starch which escapes digestion by host enzymes, is ascribed to a

number of positive physiological effects and benefit to the colon microbiota

(Annison & Topping, 1994; Ferguson et al., 2000; Topping et al., 2008). Most

of the health benefits ascribed to SDS are deduced from low-GI foods which

may have a similar glycaemic response as SDS. However, not all low-GI foods

have a slow digesting component. The property of SDS has not been studied

clinically to a significant degree.

When considering possible physiological effect/differences between

chronic consumption of foods high in RDS versus SDS, one might first

look at the level of the gut itself. RDS releases glucose quickly after leaving

the pyloric valve of the stomach, and is absorbed in the duodenum or proximal

jejunum. The exact fate of SDS is somewhat less clear. While most SDS-

containing foods still have a rapidly digesting component, certainly some

portion of the starch or its intermediate degradation products is digested and

releases glucose into the distal jejunum and ileum.

Such differences in starch digestion rate have, in recent years, been

documented to cause changes at the level of the gut enterocytes in terms

of gene expression that lead to changes in levels of the mucosal a-glucosi-dases (Mochizuki et al., 2010a, 2010b), glucose transporters (Shimada et al.,

2009) and gut hormones (Wachters-Hagedoorn et al., 2006; Shimada et al.,

2008), all of which could affect human physiology and health.

Seal et al. (2003) showed that,when fed to human subjects, normal uncooked

corn starch,which is a good source of SDS, resulted in a slowandprolonged (�5

hours) postprandial release of glucose, as well as low insulin levels throughout

Slowly Digestible Starch and Health Benefits 113

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the postprandial phase (Figure 6.1). Unlike RDS, there was a lack of a

hypoglycaemic episode with SDS ingestion and there was a shift in peak blood

glucose to a longer time. SDS consumption also extended exogenous glucose

oxidation and resulted in a lower level of plasma-free fatty acids.

In the study byWachters-Hagedoorn et al. (2006), ingestion of normal corn

starch resulted in elevated and prolonged increase in the incretin hormones

glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic poly-

peptide (GIP) – the former up to 300 minutes postprandial. Apart from its

insulin stimulation role, GLP-1 has been implicated in decreasing gastric

emptying and improvement in insulin sensitivity and glycaemic response in

diabetics. This suggests that slowly digesting carbohydrates (SDC) in general

may be beneficial on energy homeostasis and regulation. SDC may have

additional benefits in areas of cognitive/mental performance (Benton et al.,

2003; Lang et al., 2003) and satiety (Sparti et al., 2000; Araya and Alvi~na,2004). The review of Lehmann & Robin (2007) discuss these other areas of

possible benefit.

Thus, SDS is associated with positive health outcomes that may include

moderated postprandial glycaemia, circulating free fatty acids and oxidative

stress (Jenkins et al., 2002). Harbis et al. (2004) showed that slowly available

glucose, the in vivo counterpart to SDS, when provided to obese and insulin-

resistant individuals, resulted in some improved metabolic profiles, including

lower postprandial insulinaemia and circulating triacylglycerols. Moreover,

high levels of SDS-containing legumes are considered beneficial to diabetic

control (McCrory et al., 2010), and it has been shown that a bedtime meal

containing SDS (corn starch) improved glucose tolerance for the second

Figure 6.1 Depiction of slowly digesting side-by-side mechanism and resultinginside-out digestion pattern (Zhang et al., 2006a). Reprinted with permissionfrom Zhang et al., 2006a. Copyright 2006 American Chemical Society.


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morning meal (Axelsen et al., 1999). SDS-containing foods may be a dietary

intervention to curb or delay the prevalence of metabolic syndrome and

related diseases such as cardiovascular disease and diabetes (Cook et al.,

2003; Hu et al., 2004; Giugliano et al., 2006).

6.3 THE PROCESS OF STARCH DIGESTION

Starch is digested in the mammalian gastrointestinal tract first by salivary and

pancreatic a-amylases which produce small linear and branched a-limit

dextrins, which are further digested to glucose by the a-glucosidases,maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) at the mucosal

brush border of the small intestines (Swallow et al., 2001; Nichols et al., 2003).

The resulting glucose is then absorbed by the sodium-glucose active transporter

(SGLT-1) found in the apicalmembrane of the enterocytes (Pencek et al., 2002).

This is a very efficient system of reducing starch to glucose, though the rate can

be influenced by a number of intrinsic factors related to the starchy food (food

matrix, starch molecular form (dispersed, retrograded, crystalline), natural

enzyme inhibitors) (Tester et al., 2004).Gelatinized starches that are completely

dispersed are particularly susceptible to enzyme action.

A significant, though often overlooked, factor in determining the rate of

starch digestion and glucose absorption is the transit time of food through the

upper gastrointestinal tract. This is related principally to gastric emptying

time, which is controlled by gut hormones as triggered by macronutrients or

short chain fatty acids generated by fermentation of dietary fibre, including

resistant starch, as well as food form (solids vs. liquids, viscosity). Viscosity

itself can delay starch digestion by limiting access of enzymes to substrate. It

is possible that these factors can be used to generate a slow starch digestion

effect.

6.3.1 Enzyme action

Salivary and pancreatic a-amylases (E.C. 3.2.1.1) are a-1,4 endo-

glucosidases. It is generally considered that starch is only slightly digested

by salivary amylase, with the bulk of hydrolysis occurring by the action of

pancreatic a-amylase. There are five sub-sites for starch binding for human

a-amylase (Brayer et al., 2000), and cleavage of the a-1,4 glycosidic linkagesuses a multiple attack mechanism (Mazur & Nakatani, 1993) to produce

a-limit dextrins. On the other hand, the brush border a-glucosidases hydro-lyze both a-1,4 and a-1,6 glycosidic bonds and produce glucose. Regarding

kinetics of bond cleavage, a-1,6 glycosidic bonds hydrolyze more slowly


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compared to a-1,4 bonds (Kerr et al., 1951), and this approach has been usedto slow starch digestion rate (Ao et al., 2007).

While pancreatic a-amylase plays a pivotal role in starch digestion

(Englyst et al., 1992), the mucosal a-glucosidases are a recognized control

point for glucose generation and, consequently, are of equal importance in

strategies to slow glucose delivery from starch. Even the a-amylase degrada-

tion products of maltotriose, maltotetraose, and maltopentose have been

shown to have some inhibitory action on the most active of the four

a-glucosidases, the glucoamylase (Quezada-Calvillo et al., 2007, 2008).

Mucosal MGAM (E.C. 3.2.1.20 and 3.2.1.3, encoded by the gene MGAM,

located on chromosome 7q34) and SI (E.C. 3.2.1.48 and 3.2.1.10, encoded by

the gene SI, located on chromosome 3q26) (Nichols et al., 1998, 2003) are

complexes of two enzymes each that belong to the glucohydrolyase Family 31.

All four enzyme subunits have a-1,4 exo-glucosidic activity from the non-

reducing ends of maltooligosaccharides. Though commonly termed maltase,

glucoamylase, isomaltase and sucrase, these enzymes all have maltase activity

and other activities overlap into different subunits. This lack of specificity of

terminology has led to the use of terms associated with their location relative

to the anchor point on the brush border enterocytes. Thus, N-terminal MGAM

is maltase, C-terminal MGAM is glucoamylase, N-terminal SI is isomaltase

and C-terminal SI is sucrase. The main debranching enzyme is Nt-SI although

Nt-MGAM also has some isomaltase activity (Sim et al., 2010). Ct-MGAM

has higher hydrolytic property for a-glucans with longer chains (Sim et al.,

2008), and can even digest native starch amylopectin (Lin et al., 2012).

6.4 STRUCTURAL AND PHYSIOLOGICALFUNDAMENTALS OF SDS

SDS is not a defined entity of starch, as is RS with its subcategories of RS 1-4

types, and it is more of a concept than a specific material; it is an in vitro

measurement of starch digestion between 20 and 120 minutes when using the

enzymes and conditions as stated by Englyst et al. (1992). It corresponds to

the in vivo concept of slowly available glucose (SAG), although SAG can

come also from other slowly digestible glycaemic carbohydrates. Accord-

ingly, knowledge of starch structure and the various ways that SDS can be

generated is necessary to develop strategies for SDS product development. In

our view, there are four basic mechanisms for generating SDS:

1. physical or food matrix structures that decrease enzyme accessibility;

2. chemical structures that limit rate of hydrolysis;


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3. other food factors that decrease digestion rate (e.g. viscosity, inhibitors);

4. physiological control of food motility (i.e. gastric emptying).

6.4.1 Physical or food matrix structures related to SDS

Processed foods, such as pasta, often have structures related to food matrices

that lead to SDS. These are generally where starch in the food is less

accessible due to other surrounding food components, such as the dense

protein matrix of pasta (Fardet et al., 1998) or hard-to-digest protein matrix in

sorghum porridges (Zhang & Hamaker, 1998).

It is raw starch itself, with its natural semi-crystalline granular structure

comprised of concentric layers of amorphous and crystalline regions, that is

perhaps the best example of SDS physical structure. Amylopectin fine

structure differences form the basis of the semi-crystalline A, B, and C

x-ray diffraction patterns (Thompson, 2000). Due to a combination of

crystallite packing and accessibility of the granule to enzymes afforded by

surface pores leading to interior channels, raw cereal starches naturally have a

high amount of SDS material (Seal et al., 2003).

G�erard et al. (2001), Jane et al. (1997), and Planchot et al. (1997) have

shown that B-type tuber starches are resistant to enzyme digestion, while

A-type cereal starches are mostly slowly digestible. While amorphous

regions of the starch granule have been thought to be more easily digested

than crystalline regions (Gallant et al., 1992), Zhang et al. (2006a, 2006b)

revealed that degree of crystallinity did not change after amylolysis,

indicating that amorphous and crystalline regions are digested simulta-

neously. The proposed side-by-side digestion mechanism starts from the

channels (see Figure 6.1) and proceeds to the observed inside-out digestion

pattern. Apparently, this is caused by tight linkages between amorphous and

crystalline layers. The layer-by-layer crystalline (A-type) and amorphous

regions are the structural basis for the slow digestion property of native

cereal starches, providing a strategy for developing starch-based structures

that are slowly digestible.

As mentioned, supramolecular level food matrices, such as exist in cooked

pasta with protein networks, may cause slow starch digestion due to limited

accessibility of starch-degrading enzymes to starch (Fardet et al., 1998). To

better control starch digestion rates for experimental purposes, we made

starch-entrapped microspheres that consist of starch in a quick-setting gel

(sodium alginate in calcium chloride) (Venkatachalam et al., 2009; Figure

6.2). The porous polysaccharide structure permits entrance of amylase to

gelatinized starch and the rate of digestion is controlled by pore size. This

provides a strategy to achieve SDS.


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6.4.2 Starch chemical structures leading to SDS

Twomain factors regarding starch structure affect its digestion rate: the degree

and extent of retrogradation; and the amount and placement of a-1–6 branch

points. Certainly, most starchy foods that are consumed are cooked. In this

process, when water content is sufficient, starch gelatinizes, with a concomi-

tant loss in starch crystalline structure and a large increase in rate of starch

digestion. Generally cooked processed foods have very little SDS component.

Regarding retrogradation of starch, gelatinized amylose retrogrades rap-

idly and forms double helices in the range of 40 to 70 DP long that align into

higher-ordered crystalline structures that become resistant in nature (Leloup

et al., 2004). It is not clear whether a strategy of controlling amylose

retrogradation into more slowly digesting structures is possible, but this could

be a way to obtain SDS. A less well known effect of retrogradation on

digestion rate is that of amylopectin. Retrogradation of the external chains

Figure 6.2 Starch-entrapped microspheres in a gelled porous alginate matrixdigested to 120 minutes with the in vitro Englyst assay. Reproduced with permissionfrom Venkatachalam et al., 2006.


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slows rate of digestion (Zhang et al., 2008b). Along this line, retrogradation of

partially debranched amylopectin after isoamylase treatment has been used to

make SDS (Shi et al., 2003; Shin et al., 2004).

In our own investigations (Zhang et al., 2008a, 2008b), we showed that

amylopectin fine structure differences can drive the SDS amount. Using a

range of maize starch mutants, two paths towards increase were found,

represented by a parabolic relationship between SDS content and the weight

ratio of amylopectin short chains (DP< 13) to long chains (DP� 13)

(Figure 6.3). Amylopectin with either a high amount of short chains (higher

branch points and shorter external chains), or a higher proportion of long

internal chains, had a higher content of SDS (Figure 6.3).

Two mechanisms were found to drive slow digestion of gelatinized

starches: inherently high branch density somewhat slows digestion due to

the slower kinetics associated with digestion of a-1,6 linkages, while high

long chains drives faster retrogradation, as has been noted by others (Klucinec

& Thompson, 1999; Matalanis et al., 2009). In the latter retrograded type, the

SDS property appeared to be transient in nature, as increased storage time

converted some SDS to RS (Zhang et al., 2008b). Regarding the effect of

branched density, another a-glucan structure with high a-1,6 linkages, thoughnot in a branched structure like amylopectin, is pullulan, and digestion was

likewise affected (Wolf et al., 2003). Thus, amylopectin seems associated

with the SDS property.

R2 = 0.7147

Group I Group II

SF/LF weight ratio

SD

S (

%)

25

20

15

10

5

01 0.8 0.6 0.4 0.2 0

Figure 6.3 A parabolic relationship between SDS percentage and the weight ratio ofshort-chain (SF) to long-chain fraction (LF) (Zhang et al., 2008a). Reprinted withpermission from Zhang et al., 2008a. Copyright 2008 American Chemical Society.


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6.4.3 Other food factors that decrease digestion rate

Much focus on decreasing starch digestion rate has been on food viscosity, as

well as a-amylase and a-glucosidase natural inhibitors, and other factors suchas organic acids. These have been associated mostly with changes in gly-

caemic response or GI.

Water-soluble polysaccharides that impart significant viscosity effect have

been shown to result in slower digestion and absorption of glycaemic

carbohydrates, and also reduced postprandial glycaemic and insulinaemic

responses (Jenkins et al., 1976, 1977). Common fibres in this category include

b-glucans, psyllium and guar gum, and these have been shown to moderate

glycaemic response even when incorporated into food products (Wood et al.,

1990; Yokayama et al., 1997; Jenkins et al., 2002). At a mechanistic level,

viscosity appears to cause a slower gastric emptying as well as lower

accessibility of enzymes to substrates (Lecl�ere et al., 1994; Slavin, 2005),

accompanied by a reduced diffusion rate of digested carbohydrates to the

mucosal surface (Briani et al., 1987).

Natural a-amylase and a-glucosidase inhibitors can moderate starch

digestion to blunt both glycaemic and insulinaemic responses. Strong inhibi-

tion can result in an increase in resistant starch, though partial inhibition can

produce a slowly digestible effect. Acarbose, a modified tetrasaccharide

formed by the genus Actinoplanes, is one of the best studied of such inhibitors,

with most specific inhibition of the C-terminal a-glucosidases (Hiele et al.,

1992; Eskandari et al., 2011). Due to its blunting of postprandial glycaemic

response, it has been used in treatment of diabetes (Chiasson et al., 1994;

Conniff et al., 1994). A well-studied a-amylase inhibitor comes from Great

Northern beans (phaseolamin) and has been shown to promote glucose

homeostasis (Boivin et al., 1988).

There is a significant literature on natural inhibitors of both a-amylase and

a-glucosidases. A number of phenolic compounds, such as those found in green

tea extract (Konishi et al., 2006), inhibit enzyme activity and lower glycaemic

response. As a strategy to slow glycaemic carbohydrate digestion and/or absorp-

tion, use of natural inhibitors in foods necessitates more study to determine

physiologically relevant levels and other potential unintended consequences.

Lower glycaemic and insulinaemic responses have been noted with

consumption of organic acids, such as found in sourdough fermentation

(Liljeberg and Bj€orck, 1996, 1998; Liljeberg et al., 1995). For example,

sodium propionate added to bread decreased postprandial blood glucose and

insulin responses and also increased satiety compared to control breads. This

was attributed to delayed gastric emptying. Vinegar added to a starchy meal

had a similar effect (Liljeberg and Bj€orck, 1998).


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6.4.4 Physiological control of foodmotility

Slow digestion of starch can perhaps be best achieved by control of transit

time of food through the upper gastrointestinal tract, and this is mainly

through control of the rate of gastric emptying. Both the ileal break and the

colonic break have the effect of slowing gastric emptying. The former relates

to sensing of macronutrients (fat, protein, carbohydrate) in the ileum, through

triggering of gut hormones and the nervous system that causes longer

retention of food in the stomach (Van Citters & Lin, 2006). The colonic

break relates mainly to dietary fibre, including RS, fermentation that also

triggers the gut hormone peptide YYand, perhaps, GLP-1, which slows gastric

emptying (Massimino et al., 1998; Cuche et al., 2000).

Starch that is portioned out of the stomach to the small intestine over an

extended period of time is, in essence, a ‘slowly digestible starch’. Dietary fibre

fermentation also can cause the ‘second meal effect’, as noted by Jenkins et al.

(1982), Wolever et al. (1988) and Brighenti et al. (2006), which moderates

glycaemic and insulinaemic responses of the subsequent meal. Resistant starch

has specifically been shown to have this effect (Bj€orck et al., 2000).

6.5 APPLICATION-ORIENTED STRATEGIESTOMAKE SDS

Currently, there are very few approaches to make commercially viable SDS.

Practically, at this time strategies can be reduced to two areas: starch

molecular structures that reduce digestion rate; and food matrices with

slow starch digestion. A central question that remains, however, is what

degree of slow digestion effect, and what amount consumed, is necessary to

obtain a desired physiological, health-related outcome. This is one of the main

unanswered questions that impedes SDS development of commercial

products.

6.5.1 Starch-based ingredients

As mentioned above, starch amylopectin structures with high branching

and short external chains, or with a high proportion of long chains, have

higher SDS contents. Additionally, enzymatic, physical, and chemical

modifications of starch have been attempted, with the aim of decreasing

digestion rate.

Modification of starch molecular structures through genetic manipulation

of synthesizing enzymes, or through enzyme modification by processing,


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is an approach with good potential for the creation of SDS products. Main

strategies would be to produce highly branched structures with short external

chains or structures that retrograde to slowly digestible crystallites. b-amylase

has been used to shorten non-reducing external chains of amylopectin to

increase branch density and branching enzymes to add branches (Hamaker &

Han, 2006; Ao et al., 2007). The patent of Backer & Saniez (2005) showed a

method to produce a highly branched soluble a-glucan using an enzymatic

approach. Shi et al. (2003), in their patent, showed a method of making

retrograded debranched linear chains that are slowly digestible.

Physical modification of starch to slow starch digestion rate has been

achieved by heat-moisture treatment, temperature cycling and storage condi-

tions. Hydrothermal treatment of sweet potato starch resulted in A-type

crystalline structure that coincided with a large increase in SDS over native

granules (Shin et al., 2004). Using waxy potato starch, Lee et al. (2011)

increased SDS content to 42% by heat-moisture treatment and gave a

favourable glycaemic response in mice. Temperature-cycling retrogradation

of waxy rice starch also resulted in a high SDS value of 52% (Zhang et al.,

2011).

Regarding chemical modification, digestion properties of starch can be

changed through the addition of functional groups to the linear chains of

starch. Many modification methods make some degree of RS, such as

oxidation, acetylation, cross-linking and etherification (Wolf et al., 1999).

Octenyl succinic anhydride (OSA) esterification, in particular, appears to

increase SDS content (Han & BeMiller, 2007), and Wolf et al. (2001) found

modified in vivo glycaemic response of OSA-modified starch. OSA-modified

starch molecules slow enzyme digestion through an uncompetitive inhibitor

mechanism (He et al., 2008).

6.5.2 SDS generation in a food matrix

Another approach to making SDS products is through use of food processing

technologies to create matrices that slow access to starch and to limit the

extent of starch gelatinization, which also slows digestion. Dense food

matrices, such as dense pastas, are normally associated with SDS, though

the interactions of specific food components with starch can also slow starch

digestion. The most recognized interaction is the inclusion complex between

amylose and lipid.

Lower digestibility has been noted in foods that have amylose-

monoglyceride complexes (Holm et al., 1983; Seneviratne & Biliaderis,

1991; Murray et al., 1998). Saturated monoglycerides with long aliphatic

tails cause slower digestion (Eliasson & Krog, 1985). Patil et al. (1998)


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showed that amylose-lipid complexes mixed in the diet of dogs somewhat

lowered glycaemic and insulinaemic responses. Starch conjugated through the

Maillard reaction with protein or amino acids has also been shown to reduce

digestibility (Yang et al., 1998). Legume or pulses, particularly when cooked

as a whole grain, have a comparably high content of SDS that is at least

partly due to physical entrapment in cell walls (Englyst et al., 1996; McCrory

et al., 2010).

6.6 CONSIDERATIONS

There is growing evidence that the rate and location of starch digestion in the

upper gastrointestinal tract has physiological and metabolic consequences that

relate to health. This applies not only for starch, which is the major glycaemic

carbohydrate in most diets, but also for other slowly digestible carbohydrates.

While SDS is distinctly different from RS in that it is digestible and generates

glucose directly for absorption in the small intestine, starch digestion rate is

impacted by RS and its effect on gastric emptying. Additionally, glucose

release in the ileum of the small intestine also likely slows gastric emptying.

When considering the wide range of time of postprandial digestion of starches

in foods, perhaps control of gastric emptying has the largest single effect on

rate of starch digestion, and SDS may itself lengthen emptying time through

feedback mechanisms.

However, even considering that some methods exist to make SDS ingredi-

ents or foods, there is a lack of knowledge as to the amount and manner of

delivery that is needed to produce a desired effect. Further understanding of

consequences of SDS that might include energy balance, metabolic diseases,

cognitive and physical performance and satiety, are needed. New technologies

also must be identified to make robust and more defined SDS materials for use

in the food industry.

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Araya, H., Alvi~na, M. (2004). Rapid carbohydrate digestion rate produced lesser short-term satiety in obese preschool children. European Journal of Clinical Nutrition 58,637–642.


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Axelsen, M., Arvidsson, L.R., Lonnroth, P., Smith, U. (1999). Breakfast glycaemicresponse in patients with type 2 diabetes: effects of bedtime dietary carbohydrates.European Journal of Clinical Nutrition 53, 706–710.

Backer, D., Saniez, M.H. (2005). Soluble highly branched glucose polymers and theirmethod of production. US Patent 2005/0142167A1.

Benton, D., Ruffin, M., Lassel, T., Nabb, S., Messaoudi, M., Vinoy, S., Desor, D., Lang, V.(2003). The delivery rate of dietary carbohydrates affects cognitive performance in bothrats and humans. Psychopharmacology 166, 86–90.

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7 Measurement of Resistant Starch andIncorporation of Resistant Starch intoDietary Fibre Measurements

Barry V. McClearyMegazyme International, Bray Business Park, Ireland

7.1 INTRODUCTION

Resistant starch (RS) is that portion of the starch that is not broken down and

absorbed in the small intestine of humans. It enters the large intestine, where it

is partially or wholly fermented. The presence of a starch fraction resistant to

enzymic hydrolysis was first recognized by Englyst et al. (1982) during their

research on the measurement of non-starch polysaccharides.

Several in vivo approaches have been adopted for the measurement of

resistant starch, including: the hydrogen breath test; direct collection of ileal

effluent from patients (ileostomy patients) who have had the colon removed;

and direct collection of the ileal effluent from healthy subjects using a long

triple lumen tube (Champ et al., 2001). Of these, the ileostomy model is

considered to be the best, but not necessarily perfect.

It is generally accepted that any in vitro method used to measure resistant

starch must give values in line with those obtained with ileostomy patients.

Berry (1986) modified the in vitro method of Englyst et al. (1982) to mimic

physiological conditions more closely. Incubations were performed at 37 �C.Pancreatic a-amylase and pullulanase was again employed, but the initial

heating step at 100 �C was omitted. Using this method, the measured resistant

starch contents of samples were much higher than those previously obtained.

This was subsequently confirmed by Englyst & Cummings (1985, 1986, 1987)

through studies with healthy ileostomy subjects.

131




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These authors (Englyst et al. 1992) also divided RS into three classes,

namely:

� RS1: physically trapped starch as found in coarsely ground or chewed

cereals, legumes, and grains.� RS2: resistant starch granules or non-gelatinized starch granules which are

highly resistant to digestion by a-amylase until gelatinized, e.g. uncooked

potato, green banana and high-amylose starch).� RS3: retrograded starch polymers (mainly amylose), which are produced

when starch is cooled after gelatinization or during heat-moisture treatment

on annealing of starch granules (during which the starch is not gelatinized).

A fourth type of resistant starch (RS4; chemically modified starch) was

introduced by Brown et al. (1998). This starch, unlike RS1, RS2 and RS3,

contains additional chemical groups. Englyst et al. (1992) also reported on a

method for the measurement of readily digested starch (RDS), slowly digested

starch (SDS) and resistant starch (RS). In this method, resistant starch is

calculated by subtracting the sum of RDS plus SDS from total starch.

Although the method can yield useful information, it is very laborious and

gives poor reproducibility without extensive training of the analyst (Champ

et al., 2001). Accuracy is severely hampered by the fact that, with samples

containing high levels of starch with low resistant starch content, one large

analytical value is subtracted from another large value. In fact, the errors in the

measurement may be as large as the resistant starch value, e.g. materials with

approximately 70% starch and 2% resistant starch.

By the early 1990s, the physiological significance of RS was fully realized.

Several new or modified methods for its measurement were developed during

the European Research Program, EURESTA (Englyst et al., 1992; Champ,

1992). The Champ (1992) method, was based on modifications to the method

of Berry (1986), and gave a direct measurement of RS. Basically, sample size

was increased from 10mg to 100mg, the sample was digested with pancreatic

a-amylase only, and incubations were performed at pH 6.9 (pH 5.2 was used

by Englyst et al. (1982, 1992) and Berry (1986)). RS determinations were

performed directly on the pellet.

Muir & O’Dea (1992) developed a procedure for RS in which samples

were chewed, treated with pepsin and then with a mixture of pancreatic

a-amylase and amyloglucosidase (AMG) in a shaking water bath at pH 5.0,

37 �C for 15 hours. The residual pellet (containing RS) was recovered

by centrifugation and washed with acetate buffer by centrifugation, and the

RS was digested by a combination of heat, DMSO and thermostable

a-amylase treatments.


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Other methods for RS determination were developed by Faisant et al.

(1995), Goni et al. (1996), Akerberg et al. (1998) and Champ et al. (1999).

These modifications included changes in enzyme concentrations employed,

types of enzymes used (all used pancreatic a-amylase, but pullulanase was

removed, and in some cases replaced by AMG), sample pre-treatment

(chewing), pH of incubation and the addition (or not) of ethanol after the

a-amylase incubation step (Champ et al., 1999).

7.2 DEVELOPMENT OF AOAC OFFICIAL METHOD2002.02

While significant steps were made in the development of in vitro methods for

the measurement of resistant starch during the 1990s, none of these methods

were successfully subjected to interlaboratory evaluation. This prompted

McCleary & Monaghan (2002) to look at each of these methods in detail,

to evaluate all of the parameters involved and to identify sources of variability.

The ultimate aim was to develop a procedure that gave values in line with

those obtained with ileostomy patients, but also a method that could survive

the rigors of interlaboratory evaluation. Parameters investigated included:

a) incubation conditions (shaking/stirring, pH, temperature, time);

b) level of pancreatic a-amylase employed;

c) level of AMG employed;

d) the importance of protease pre-treatment;

e) procedures for recovery of resistant starch;

f) method for the dissolution of RS; and

g) glucose determination procedure.

Incubations were performed at physiological temperature (37 �C) in both ashaking water bath and in an arrangement in which the contents of the tubes

were continually stirred at different speeds. Incubations were allowed to

proceed for up to 24 hours, and the RS values obtained for a set of samples

were compared to values obtained from ileostomy studies. Typical results

obtained for regular maize starch (RMS) and high-amylose maize starch

(HAMS) are shown in Figure 7.1.

These incubations were performed in the presence of optimal levels of

AMG and at a pH of 6.0. This pH was chosen as a compromise to allow for the

different pH optima of a-amylase (pH 6.9) and AMG (pH 4.5) and the

determined stability of the two enzymes on extended incubation at different

pH values. At pH 6.0, pancreatic a-amylase has approximately 80% of the

Measurement of Resistant Starch and Incorporation of Resistant Starch 133

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activity at the optimal pH of 6.9, and AMG has 20% of the activity at the

optimal pH of 4.5. The level of each enzyme is adjusted to optimize the assay.

AMG is very stable at both pH 6.0 and 6.9, losing less than 5% of initial

activity on incubation at 37 �C for 16 hours. Pancreatic a-amylase is less

stable, however; even at pH 6.0, approximately 30% of activity remains on

incubation of the enzyme under assay conditions of 16 hours at 37 �C(compared to 50% at pH 6.9) (Figure 7.2).

There was considerable flexibility in the concentration of pancreatic

a-amylase used. RS values obtained for RMS and HAMS varied little,

with enzyme concentrations ranging from 15–60 units/ml of incubation

mixture (see Figure 7.1).

AMG in the incubation mixture had a considerable effect on the deter-

mined RS values (McCleary &Monaghan, 2002); this is considered to be due

to the known inhibitory effect of maltose on pancreatic a-amylase. The AMG

removed the maltose by hydrolyzing it to glucose, which has no inhibitory

effect. The effect of protease on determined RS values was studied by

including a pre-treatment with pepsin at pH 2. Results obtained indicated

that the protease pre-treatment had no significant effect on determined RS

values (McCleary & Monaghan, 2002). This may be due, in part, to the

presence of an active protease in the pancreatic a-amylase preparation used.

Figure 7.1 The effect of the concentration of pancreatic a-amylase and incubationtime on the determined RS value of regular maize starch (RMS) and high-amylosemaize starch (HAMS) at pH 6.0 and 37 �C for up to 25 hours. HAMS with pancreatica-amylase concentrations of (black triangle) 15, (black circle) 30 and (black square)60 U/mL; RMS with pancreatic a-amylase concentrations of (open triangle) 15,(open circle) 30 and (open square) 60 U/mL.


3GCH07 07/20/2013 8:10:28 Page 135

Resistant starch is crystalline and is difficult to dissolve. Solvents used to

dissolve this material include dimethyl sulphoxide (DMSO) and 2–4M

potassium hydroxide. For all samples studied, the RS was completely dis-

solved by stirring the RS containing pellet in 2MKOH in an ice/water bath for

20 minutes (conditions used by several authors). On neutralization, it was

essential to hydrolyze this starch rapidly to avoid re-crystallization (which

would again render the starch resistant to hydrolysis by AMG. To achieve this

and to simplify neutralization, a concentrated sodium acetate buffer (1.2M,

pH 3.8) was added, followed immediately by AMG (320 units/test; one unit of

AMG activity is the amount of enzyme required to release one micromole of

glucose from soluble starch per minute at pH 4.5 and 40 �C).This method for the measurement of RS was subjected to evaluation under

the auspices of AOAC International and AACC International, to determine the

interlaboratory performance statistics. The materials used in the study repre-

sented food materials containing RS (cooked kidney beans, green banana and

cornflakes) and a range of commercial starches, most of which naturally

contain (or were processed to contain) elevated RS levels. Thirty-seven

laboratories tested eight pairs of blind duplicate starch or plant material

samples, with RS values between 0.6 (regular maize starch) and 64% (fresh

weight basis). For samples excluding regular maize starch, RSDr values

Figure 7.2 Stability of pancreatic a-amylase and AMG on incubation at pH 6.0 and6.9 and 37 �C for up to 25 hours. AMG at pH 6.0 (closed triangle) and 6.9 (opentriangle); pancreatic a-amylase at pH 6.0 (open circle) and 6.9 (closed circle).


3GCH07 07/20/2013 8:10:28 Page 136

ranged from 1.97–4.2% and RSDR values ranged from 4.58–10.9%. The range

of applicability of the test is 2–64% RS.

The method was not suitable for samples with less than 1%RS (e.g. regular

maize starch; 0.6% RS). For such samples, RSDr and RSDR values are

unacceptably high. On the basis of this evaluation, the method was accepted

asAOACOfficialMethod 2002.02 andAACCRecommendedmethod 32–40.01

(McCleary et al., 2002).

7.3 DEVELOPMENT OF AN INTEGRATED PROCEDUREFOR THE MEASUREMENT OF TOTAL DIETARY FIBRE

Hipsley (1953) coined the term dietary fibre to cover the non-digestible

constituents of plants that make up the plant cell wall (known to include

cellulose, hemicellulose and lignin) with the aim of defining some property of

the constituent of the food that could be related to physiological behaviour in

the human small intestine. This definition was broadened by Trowell et al.

(1976) to become primarily a physiological definition, based on edibility and

resistance to digestion in the human small intestine. Thus, the definition

included indigestible polysaccharides such as gums, modified celluloses,

mucilages and pectin, and non-digestible oligosaccharides (NDO).

Methods which were developed to meet this analytical requirement

focused on the use of enzymes to remove starch and protein. The enzymes

employed require a defined level of activity and must be devoid of contami-

nating enzymes active on dietary fibre components such as pectin, b-glucan,arabinoxylan and other hemicelluloses. Following extensive international

collaboration, the method that evolved was AOAC Official Method 985.29,

‘Total dietary fibre in foods; enzymatic-gravimetric method’ (Prosky et al.,

1985, 1994) This method was subsequently extended to allowmeasurement of

total dietary fibre (TDF), soluble dietary fibre (SDF) and insoluble dietary

fibre (IDF) in foods (AOAC Official Method 991.43) (Lee et al., 1992) Other

modifications to these methods for fibre analysis have also been approved by

AOAC International (Theander & Aman, 1982).

In concurrent research in the UK, methods were developed for the measure-

ment of non-starch polysaccharides (NSP)(Englyst et al., 1982; Englyst &

Cummings, 1984, 1985; Englyst & Hudson, 1987), based on the original work

of Southgate (1969) and Southgate et al. (1978). These NSP procedures

measure only NSP; RS and NDO are excluded. Starch in the sample is

completely dissolved in hot dimethyl sulphoxide (DMSO), diluted in buffer

and depolymerized with thermostable a-amylase, followed by a mixture of


3GCH07 07/20/2013 8:10:28 Page 137

pancreatin and pullulanase. The NSP recovered is acid hydrolyzed to

monosaccharides, which are measured by high-performance liquid chroma-

tography (HPLC), gas-liquid chromatography (after derivatization) (Englyst

& Cummings, 1984) or colorimetrically (Englyst & Hudson, 1987). These

methods have not been successfully subjected to international interlabor-

atory evaluation.

A survey of scientists initiated in 1993 (Lee & Prosky, 1995) showed that

65% of the respondents favoured the inclusion of NDO and 80% favoured

inclusion of RS in the definition of dietary fibre. This led to the development of

methods for measurement of RS (AOAC Method 2002.02) and for a number

of NDO, including fructo-oligosaccharides (AOAC Methods 997.08 and

999.03), polydextrose (AOAC Method 2000.11), resistant maltodextrins

(AOAC Method 2001.03), and galacto-oligosaccharides (AOAC Method

2001.02).

In 1998, the American Association of Cereal Chemists began a critical

review of the current state of dietary fibre science, including consideration

of the state of the dietary fibre definition. Over the course of the following

year, the committee held three workshops and provided an international

website, available to all Web users worldwide, to receive comments. All

interested parties were provided with additional opportunity for comment.

After due deliberation, an updated definition of dietary fibre was delivered to

the AACC Board of Directors for adoption in early 2000 and published

(Anon, 2001) namely:

‘Dietary fibre is the edible parts of plants or analogous carbohydrates that are

resistant to digestion and absorption in the human small intestine with

complete or partial fermentation in the large intestine. Dietary fibre includes

polysaccharides, oligosaccharides, lignin, and associated plant substances.

Dietary fibres promote beneficial physiological effects including laxation, and/

or blood cholesterol attenuation, and/or blood glucose attenuation.’

Several definitions of dietary fibre have appeared over the past ten years.

The Food Nutrition Board of the Institute of Medicine of the National

Academies (USA) (2002) defined dietary fibre as follows: ‘Dietary fibre

consists of nondigestible carbohydrates and lignin that are intrinsic and

intact in plants. Added fibre consists of isolated, nondigestible carbohydrates

that have beneficial physiological effects in humans. Total fibre is the sum of

dietary fibre and added fibre.’

At the 30th session of the CODEX Committee on Nutrition and Foods for

Special Dietary Uses (CCNFSDU; 2008), the Committee agreed on the

following definition for dietary fibre:


3GCH07 07/20/2013 8:10:28 Page 138

‘Dietary fibre is carbohydrate polymers1 with ten or more monomeric units,2

which are not hydrolyzed by the endogenous enzymes in the small intestine of

humans and belong to the following categories:

� Edible carbohydrate polymers naturally occurring in the food as consumed.� Carbohydrate polymers which have been obtained from food rawmaterial by

physical, enzymatic or chemical means and which have been shown to have a

physiological effect of benefit to health as demonstrated by generally

accepted scientific evidence to competent authorities,� Synthetic carbohydrate polymers which have been shown to have a physio-

logical effect of benefit to health as demonstrated by generally accepted

scientific evidence to competent authorities.

The fact that a single method to measure all dietary fibre components is

needed has been known for some time. While it is possible to measure many

individual fibre components with specific and non-specific methods, total

dietary fibre cannot simply be calculated by adding the values for these

specific components to the determined value of high molecular weight dietary

fibre, as measured with AOACOfficial Methods 985.29 or 991.43. Since these

latter methods also measure some of the RS and various NDO in food

materials, summation leads to ‘double counting’ of this material (Figure 7.3;

McCleary et al., 2009).

An integrated method for the measurement of total dietary fibre was

published in 2007 (McCleary, 2007). This method allows the accurate

measurement of total high molecular weight dietary fibre (HMWDF), which

includes IDF (including RS) and higher molecular weight soluble dietary fibre

which precipitates in the presence of 76% aqueous ethanol (SDFP), as well as

lower molecular weight soluble dietary fibre which remains soluble in the

presence of 76% aqueous ethanol (SDFS). Details of this procedure are

outlined in Figure 7.4.

1When derived from a plant origin, dietary fibre may include fractions of lignin and/or other

compounds when associated with polysaccharides in the plant cell walls and if these compounds

are quantified by the AOAC gravimetric analytical method for dietary fibre analysis: Fractions

of lignin and the other compounds (proteic fractions, phenolic compounds, waxes, saponins,

phytates, cutin, phytosterols, etc.) intimately ‘associated’ with plant polysaccharides in the

AOAC 991.43 method. These substances are included in the definition of fibre insofar as they are

actually associated with the poly- or oligo-saccharidic fraction of fibre. However when

extracted or even re-introduced in to a food containing non digestible polysaccharides,

then they cannot be defined as dietary fibre. When combined with polysaccharides, these

associated substances may provide additional beneficial effects.2Decision on whether to include carbohydrates of 3 to 9 monomeric units should be left up to

national authorities.’


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The use of pancreatic a-amylase more closely simulates digestion in the

human digestive tract and yields RS values in line with those obtained with

AOACOfficial Method 2002.02, and with results from ileostomy patients. For

most food and ingredient samples analyzed, the RS values obtained with

AOAC Method 2009.01 were higher than those obtained with AOAC Method

985.29. The notable exception is for phosphate cross-linked starch (RS4),

where values obtained with Method 2009.01 are much lower than those

obtained with Method 985.29. The physiological significance of these results

will be discussed separately (McCleary et al., 2013).

This method was successfully subjected to interlaboratory evaluation

(McCleary et al., 2009) and accepted as AOAC Method 2009.01. In this

study, total HMWDF and SDFS were measured.

In an AOACI/AACCI interlaboratory study recently completed, the method

was evaluated for the measurement of IDF, SDFP and SDFS. IDF and SDFP

are measured using the standard gravimetric procedures, with allowance for

ash and non-digested protein. SDFS is analyzed by HPLC using D-sorbitol as

internal standard and a Waters Corporation Sugar Pak1 chromatographic

column. To obtain resistant starch values in line with those obtained in vivo

with ileostomy patients, incubations are performed either in a shaking water

Figure 7.3 Schematic representation of dietary fibre components measured, and notmeasured, by AOAC Official Methods 985.29 and 991.43. Also depicted are theproblems of partial measurement of RS, Polydextrosej and resistant maltodextrins bycurrent AOAC total dietary fibre methods. Most of the SDFS (galactooligosacchar-ides, fructooligosaccharides, etc) are not measured. The currently describedintegrated total dietary fibre procedure measures all components shown, with nodouble counting.


3GCH07 07/20/2013 8:10:29 Page 140

bath in orbital motion, or with a VarioMag1 magnetic stirrer at 37 �C for

16 hours. The pH is adjusted, and a-amylase and AMG are inactivated and

protein denatured by heating the sample to about 100 �C. Incubation with

protease is followed by pH adjustment and filtration to separate IDF and

soluble fibres. SDFP is precipitated with ethanol and recovered and dried.

After weighing, IDF and SDFP residues are analyzed for residual protein

and ash. The SDFS fraction (in the ethanolic filtrate) is concentrated by

rotary evaporation, re-dissolved and adjusted to pH 4.2–4.7 and incubated

with AMG to remove completely any traces of higher molecular weight

Add 40 ml of 50 mM Na maleate buffer, pH 6.0 (+ CaCl2) Containing pancreatic α-amylase + amyloglucosidase

Sample (1.00 g) in sealed 250 ml Duran bottle (in duplicate)

Incubate in shaking water bath at 37°C for 16 hours

Add 3.0 ml 0.75 M Trizma base to adjust pH to ≈ 8.2

Incubate at > 90°C for 20 min. Cool to ≈ 60°C

Add 0.1 ml protease

Incubate at 60°C for 30 min. Cool to room temperature

Add 4.0 ml of 2 M acetic acid (to adjust pH to ≈4.5) + 1 ml internal standard

Remove 1 ml for available CHO determination

Add 4 volumes of ethanol, stir, store at room temp for 1 hour, then filter

HMWDF determination LMWSDF determination

Figure 7.4 Schematic representation of the integrated TDF assay procedure, alsoshowingwhere samples canbe removed for determination of available carbohydrates.


3GCH07 07/20/2013 8:10:29 Page 141

maltodextrins. The solution is then either desalted by passage through a

column of mixed cation and anion ion exchange resins (Figure 7.5) or,

alternatively, is desalted on line using a de-ashing pre-column.

Quantitation is greatly simplified by including an internal standard. The

preferred internal standard is D-sorbitol and this is added to the sample just

prior to adding ethanol to precipitate the SDFP. A number of compounds were

evaluated as potential internal standards, including 1,5-pentanediol, dieth-

ylene glycol and triethylene glycol. Of these, diethylene glycol appeared best.

However, on closer study, some of this is lost when the SDFS fraction is rotary

evaporated, presumably by adsorbing to the glass rotary evaporator flask. This

was not observed with D-sorbitol. When D-sorbitol is rotary evaporated with a

range of sugars and NDO, the ratio of the components remains the same.

Results of this interlaboratory study have been published (McCleary et al.,

2012).

A major advantage of the described method for the measurement of total

dietary fibre is that it allows the separate measurement of IDF, SDFP and

SDFS. There is some international debate as to whether NDO (SDFS) should

be included in the dietary fibre measurements. Until there is agreement, this

Figure 7.5 Arrangement for the deionization of samples using amixed bed resin in aBio-Rad, Econo-Pacj disposable chromatographic column (cat. No. 732–1010) withan Alltech One-Way Stopcock (cat. No. 211524). Also shown is a Gilson Minipulsj

Evolution pump and collection bottles.


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oligosaccharide material can be measured and simply reported as NDO. With

minor modification, the method can also be adapted to measure digestible

carbohydrates (fructose, glucose, sucrose, maltodextrins and non-resistant

starch, and the glucose component of lactose; McCleary, 2007).

REFERENCES

Akerberg, K.E., Liljberg, G.M. Granfeldt, Y.E., Drews, A.W., Bjorck M.E. (1998). An in-vitro method based on chewing, to predict resistant starch content in foods allowsparallel determination of potentially available starch and dietary fiber. Journal ofNutrition 128, 651–660.

Anon (2001) The definition of dietary fiber. Cereal Foods World 46, 112–126.Berry, C.S. (1986). Resistant starch: formation and measurement of starch that survives

exhaustive digestion with amylolytic enzymes during the determination of dietary fibre.Journal of Cereal Science 4, 301–314.

Brown, I.L., Wang, X., Topping, D.L., Playne, M.J., Conway, P.L. (1998). High amylosemaize starch as a versatile prebiotic for use with probiotic bacteria. Food Australia 50,603–622.

Champ, M. (1992). Determination of resistant starch in foods and food products:interlaboratory study. European Journal of Clinical Nutrition 46(Suppl. 2), S51–S62.

Champ, M., Martin, L., Noah, L., M. Gratas. (1999). Analytical methods for resistantstarch. In: Cho, S.S., Prosky, L., Dreher, M. (eds). Complex Carbohydrates in Foods,169–187. Marcel Dekker, Inc., New York, USA.

Champ, M., Kozlowski, F., Lecannu, G. (2001). In-vivo and In-vitro methods for resistantstarch measurement. In: McCleary, B.V., Prosky, L. (eds). Advanced Dietary FibreTechnology106–119. Blackwell Science Ltd., Oxford, UK.

Codex Committee on Nutrition and Foods for Special Dietary Uses 28th Session. ChiangMai, Thailand, 30th October–3rd November 2006.

Englyst, H.N., Cummings, J.H. (1984). Simplified method for the measurement of non-starch polysaccharides by gas-liquid chromatography of constituent sugars as alditolacetates. Analyst 109, 937–942.

Englyst, H.N., Cummings, J.H. (1985). Digestion of the polysaccharides of some cerealfoods in the human small intestine. American Journal of Clinical Nutrition 42,778–787.

Englyst, H.N., Cummings, J.H. (1986). Digestion of the carbohydrates of banana (Musaparadisiaca sapientum) in the human small intestine. American Journal of ClinicalNutrition 44, 42–50.

Englyst, H.N., Cummings, J.H. (1987). Digestion of polysaccharides of potato in the smallintestine of man. American Journal of Clinical Nutrition 45, 423–431.

Englyst, H.N., Hudson, G.J. (1987). Colorimetric method for the routine measurement ofdietary fibre as non-starch polysaccharides. A comparison with gas-liquid chromatog-raphy. Food Chemistry 24, 63–67.

Englyst, H.N., Wiggins, H.L., Cummings, J.H. (1982). Determination of the non-starchpolysaccharides in plant foods by gas-liquid chromatography of constituent sugars asalditol acetates. Analyst 107, 307–318.


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Faisant, F., Planchot, V., Kozlowski, F., Pacouret, P., Colonna, P., Champ, M. (1995).Resistant starch determination adapted to products containing high levels of resistantstarch. Sciences des Aliments 15, 83–89.

Goni, I., Manas, E., Garcia-Diz, F. Saura-Calixto, F. (1996). Analysis of resistant starch: amethod for food and food products. Food Chemistry 56, 445–449.

Hipsley, E.H. (1953). Dietary fibre and pregnancy toxaemia. British Medical Journal 2,420–422.

Horwitz, W. (ed). (2002). Official Methods of analysis of AOAC International, 17thEdition. The Association, Gaithersberg, USA.

Institute of Medicine (2002). In:Dietary reference intakes: Energy, Carbohydrates, Fiber,Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. National Academies Press,Washington, DC.

Lee, S.C., Prosky, L. (1995). International survey on dietary fiber definition, analysis andreference materials. Journal of the Association of Official Analytical Chemists 78, 22–36.

Lee, S.C., Prosky, L., DeVries, J.W. (1992). Determination of total, soluble, and insolubledietary fiber in foods – Enzymatic-gravimetric method,MES-TRIS buffer: Collaborativestudy. Journal of the Association of Official Analytical Chemists 75, 395–416.

McCleary, B.V. (2007). An integrated procedure for the measurement of total dietary fibre(including resistant starch), non-digestible oligosaccharides and available carbohy-drates. Analytical Bioanalytical Chemistry 389, 291–308.

McCleary, B.V., Sloane, N., Draga, A., Lazewska, I. (2013) Cereal Chemistry, in press.McCleary, B.V., Monaghan, D.A. (2002). Measurement of resistant starch. Journal of theAssociation of Official Analytical Chemists 85, 665–675.

McCleary, B.V., Rossiter, P. (2004). Measurement of novel dietary fibres. Journal of theAssociation of Official Analytical Chemists 87, 707–717.

McCleary, B.V., McNally, M., Rossiter, P. (2002). Measurement of resistant starch byenzymic digestion in starch samples and selected plant materials: Collaborative Study.Journal of the Association of Official Analytical Chemists 85, 1103–1111.

McCleary, B.V., Mills, C., Draga, A. (2009). Development and evaluation of an integratedmethod for the measurement of total dietary fibre. Quality Assurance and Safety ofCrops and Foods 1, 213–224.

McCleary, B.V., DeVries, J.W., Rader, J.I., Cohen, G., Prosky, L., Mugford, D.A., Champ,M., Okuma, K. (2010). Determination of total dietary fiber (CODEX definition) byenzymatic-gravimetric method and liquid chromatography: collaborative study. Journalof the Association of Official Analytical Chemists 93, 221–33.

McCleary, B.V., DeVries, J.W., Rader, J.I., Cohen, G., Prosky, L, Mugford, D.C., Okuma,K. (2012). Determination of insoluble, soluble, and total dietary fiber (CODEX defini-tion) by enzymatic-gravimetric method and liquid chromatography: collaborative study.Journal of AOAC International 95, 824–844.

Muir, J.A., O’Dea, K. (1992). Measurement of resistant starch: Factors affecting theamount of starch escaping digestion in vitro. American Journal of Clinical Nutrition56, 123–127.

Prosky, L., Asp, N-G., Furda I, DeVries, J.W., Schweizer, T.F., Harland, B.F. (1985).Determination of total dietary fiber in food and food products: collaborative study.Journal of the Association of Official Analytical Chemists 68, 677–679.


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Prosky, L., Asp, N-G., Schweizer, T.F. DeVries, J.W., Furda, Lee, S.C. (1994). Determi-nation of soluble dietary fiber in foods and food products: Collaborative study. Journal ofthe Association of Official Analytical Chemists 77, 690–694.

Southgate, D.A.T. (1969). Determination of carbohydrates in foods. II. Unavailablecarbohydrates. Journal of the Science of Food and Agriculture 20, 326–330.

Southgate, D.A.T., Hudson, G.J., Englyst, H.N. (1978). The analysis of dietary fibre, thechoices for the analyst. Journal of the Science of Food and Agriculture 29, 979–988.

Theander, O., Aman, P. (1982). Studies on dietary fibre: A method for the analysis andchemical characterization of total dietary fibres. Swedish Journal of AgriculturalResearch 9 (3), 97–106.

Trowell, H.C., Southgate, D.A.T., Wolever, T.M.S., Leeds A.R., Gassull, M.A., Jenkins,D.J.A. (1976). Dietary fiber redefined. Lancet 1, 967.


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8 In Vitro Enzymatic Testing Methodand Digestion Mechanism ofCross-linked Wheat Starch

Radhiah Shukri,1 Paul A. Seib,1 Clodualdo C.Maningat,1,2 and Yong-Cheng Shi1

1Department of Grain Science and Industry, Kansas State University, USA2MGP Ingredients, Inc., USA

8.1 INTRODUCTION

Early studies have shown that a fraction of some starches consumed by

humans escape the small intestine (Anderson et al., 1981; Stephen et al.,

1981; Englyst & Cummings, 1985; Asp et al., 1987). The total amount of

starch and products of starch degradation that resist digestion in the small

intestine of healthy people is termed resistant starch (RS) (Asp, 1992). RS is

further categorized into five classes: physically inaccessible starch known as

RS1; granular starch known as RS2; cooked and retrograded starches known

as RS3; chemically modified starches known as RS4; and amylose-lipid

complex (Brown et al., 2006; Hasjim et al. 2010). The degree of starch

digestibility is affected by the structure of starch granules, the physical

characteristics of food, the amylose and amylopectin ratio, retrogradation

of amylose and the presence of other nutrients and anti-nutrients (Sharma

et al., 2008; Bird et al., 2009).

Chemical modification of starch has been proved to affect the extent and

rate of digestibility in the small intestine (Wolf et al., 1999), based on starch

source, the type and degree of modification, extent of granule gelatinization

and the source of enzyme used (Filer, 1971). Cross-linked (CL) starch is

one of the most highly produced and utilized chemically modified starches in

the food industry (Wurzburg, 1986). The highest cross-linking levels that are

acceptable as food starch with 0.4% phosphorus (P) add-ons are achievable by

145




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using 12% sodium trimetaphosphate (STMP) and sodium tripolyphosphate

(STPP) as phosphorylating agents (Woo & Seib, 2002).

Several in vitro methods have been suggested to quantify RS in food,

all of which involve enzymatic digestions on starch, removal of the

digested starch and quantitation of remaining RS. Quantitation of RS can

be carried out using direct methods or indirect methods. While direct

methods determine RS in the remaining starch after removing the

digested portion (Berry, 1986; Champ, 1992; Faisant et al., 1993;

McCleary et al., 2002), the indirect methods measure RS by subtracting

the digested starch from the total starch (Englyst, et al., 1992; Tovar

et al., 1990). Determination of RS is achieved by solubilizing RS and

quantifying with colorimetric methods (Berry, 1986; McCleary et al.,

2002), or by isolating RS gravimetrically after ethanol precipitation

(Lee et al., 1992; McCleary et al., 2010).

Most methods use a-amylase (Englyst et al., 1992) or a cocktail of

a-amylase and amyloglucosidase (McCleary et al., 2002, 2010) to hydrolyze

the RDS and SDS fractions. Enzyme cocktails are used to avoid possible

inhibition of a-amylase by maltose and maltotriose (Sharma et al., 2008). The

extrinsic factor (i.e., extent of chewing, rate of orocecal transit), incubation

temperature, incubation time and enzyme source may affect the in vitro yield

of these assays (Englyst et al., 1992).

Table 8.1 shows a comparison of in vivo and in vitro RS content using

Englyst, AOAC Method 991.43, AOAC Method 2002.02 and AOAC

Method 2009.01 in several samples. The RS content of each sample either

differed slightly or significantly when compared to the in vitro methods

used. Several samples determined by the Englyst method (raw potato

starch, cornflakes and raw green banana) and AOAC Method 2002.02

(raw potato starch and raw green banana) showed results consistent with the

in vivo data. Although AOAC Method 991.43 was reported to have

consistency with ileostomy patients fed with bean and potato flakes

(Schweizer et al., 1990), the RS reported in Table 8.1 revealed that

none of the samples except cornflakes had similar in vivo compatibility

with the RS determined by AOAC Method 991.43. The significantly lower

RS (determined by AOAC Method 991.43) of raw potato starch, high-

amylose corn starch and raw green banana was likely due to a higher

incubation temperature (100 �C).The most current in vitro method, namely AOAC Method 2009.01, claims

to effectively determine non-starch polysaccharides, RS and non-digestible

oligosaccharides content in samples (McCleary et al., 2010). The latter

method applies key features of AOAC Method 985.29, AOAC Method

991.43, AOAC Method 2001.03 and AOAC Method 2002.02 (McCleary


CH08GR 07/29/2013 16:22:48 Page 147

et al., 2010). The starch digestion conditions in AOAC Method 2009.01 were

similar to those used in AOACMethod 2002.02, which involve the incubation

of a sample with a pancreatic a-amylase and amyloglucosidase cocktail for 16

hours at 37 �C. Although the digestive enzyme concentration in AOAC

Method 2009.01 was significantly higher, results produced by AOACMethod

2009.01 and AOAC Method 2002.02 were claimed to be similar for most

samples (McCleary, 2007).

The RS content by AOAC Method 2009.01 in the samples listed in Table

8.1 showed no consistency with in vivo data. Based on a comparison of in vitro

methods (Englyst Method, AOAC Method 991.43, AOAC Method 2009.01)

on CLwheat starch, AOACMethod 2009.01 provided a significantly lower RS

content (Table 8.1). AOAC Method 2002.02 was not included in the latter

comparison, because CL wheat starch is not able to solubilize in 2M

potassium hydroxide for determination of glucose content using the colori-

metric procedure.

The findings of RS content in CL wheat starch raise questions as to the

efficiency of AOAC Method 2009.01 to quantify RS and the compatibility

with in vivo results. As opposed to the Englyst Method, AOAC Method

2009.01 differs in enzyme concentration, buffers, measurement method,

incubation time and enzyme sources, but both methods employ the same

Table 8.1 Comparison of resistant starch (RS: % total starch) content in raw potatostarch, high-amylose corn starch, corn flakes and raw green banana determined byin vitro methods (Englyst Method, AOAC Method 991.43, AOAC Method 2002.02 andAOAC Method 2009.01) and in vivo method (ileostomy model).

In vitro RS (%)

Source of starch EnglystAOAC991.43

AOAC2002.02

AOAC2009.01

In vivoRS (%)

Raw potato starch 66.5a 0.9b 64.9b 56.8b 67.9c

High-amylose corn starch 71.4a 25.6b 50.0b 49.3b 43.7c

Corn flakes 3.9a 3.3e 2.2b 2.4b 3.1–5.0d

Raw green banana 54.2b 7.5b 51b 38b 55.3c

Cross-linked wheat starch� 81.7 82.3 –�� 23.9 –

aEnglyst et al. (1992).bMcCleary (2007).cLangkilde & Andersson (1995).dMuir and O’Dea, (1992).ePendlington (1999).� RS content was determined by in vitro methods in Kansas State University lab.�� Unable to determine RS in cross-linked wheat starch due to insolubility of cross-linked starch in 2M

potassium hydroxide.

In Vitro Enzymatic Testing Method and Digestion Mechanism of Cross-linked 147

CH08GR 07/29/2013 16:22:48 Page 148

incubation temperature (37 �C). The digestion kinetics, specifically the

mechanism of enzyme action and incubation time in AOAC Method

2009.01, was our major interest. We predicted that 16 hours of incubation

time, with a relatively high amount and concentration of digestive enzyme

(40ml a-amylase (50U/ml) and amyloglucosidase (3.4U/ml)), would be too

harsh of a condition for CLwheat starch. Hence, the objectives of this research

were to study the in vitro digestion behaviour of CL wheat starch at various

time intervals, the progressive changes of CL wheat starch granules during the

in vitro digestion and the mechanism of the digestive enzymes during the

digestion period in AOAC Method 2009.01.

8.2 MATERIALS ANDMETHODS

8.2.1 Materials

CL wheat starch (Fibersym1 RW) and native wheat starch (MidsolTM 50)

were obtained from MGP Ingredients, Inc1 (Atchison, Kansas). The inte-

grated total dietary fibre assay kit (catalogue no. INTDF 06/12) was purchased

from Megazyme International Ireland Ltd. (Wicklow, Ireland). All chemicals

were reagent grade.

8.2.2 General methods

P content was assayed using the procedure of Smith & Caruso (1964.)

Moisture content was obtained according to AACC Method 44-15 (AACC

2000).

8.2.3 Conversion of CL wheat starch to phosphodextrinsand 31PNMR spectra of the phosphodextrins

The treatment was based on the method described by Sang et al. (2010).

Starch (1.0 g db) was weighed into a 50ml centrifuge tube. The starch was

slurried with 30ml 2.0mM calcium chloride at pH 8.2, and heat-stable

a-amylase (100ml) was then added. The mixture was heated in a boiling

water bath for 30 minutes and 100ml of heat stable a-amylase was added into

the mixture again. After cooling to room temperature, the pH of the mixture

was adjusted to 4.5, and amyloglucosidase (200ml) was incorporated for one

hour to incubate at 60 �C. The mixture was adjusted to pH 7.0, centrifuged

(1500� g, 10min), and the supernatant was freeze-dried.


CH08GR 07/29/2013 16:22:48 Page 149

The freeze-dried starch digest (1.0 g, db) was dissolved in deuterium

oxide (1.0ml) containing 20mM EDTA and 0.002% sodium azide. The pH

of the solution was adjusted to 8.0 by adding 0.1M sodium hydroxide. The

proton-decoupled 31P NMR spectra were obtained using a 11.75 Tesla

Varian NMR System (Varian Inc., Palo Alto, CA). Using a method by Sang

et al. (2007), the 1H NMR was operated at 499.84MHz and 31P at

202.34MHz. The 31P NMR data collection was carried out at 25 �C using

a delay of six seconds between pulses, with a pulse width of 15.0ms and a

sweep width at 12,730Hz. The samples were run for eight hours for 31P

spectra detection. The obtained spectra were processed and analyzed using

Varian software VNMRJ Version 2.2C. Chemical shifts were reported in d(ppm) from the reference signal of an 85% phosphoric acid external

standard.

8.2.4 Digestibility of CL wheat starch

Digested starch of native and CL wheat starches were determined at 0, 1, 2, 4,

6, 8, 16 and 24 hours, using the a-amylase and amyloglucosidase incubation

procedure from AOAC Method 2009.01. To collect undigested starch for

characterization, CL wheat starch was weighed to 1.0 g (db), wet with ethanol

(1.0ml), and a pancreatic a-amylase/amyloglucosidase mixture (40ml) was

added to a 250ml glass bottle. The pancreatic a-amylase/amyloglucosidase

mixture contained 50 units (U)/ml of a-amylase and 3.4U/ml amylogluco-

sidase. 1 U of a-amylase at pH 5.8 and 37 �C was defined as the amount of

enzyme required to release one micromole of D-glucose per minute from

soluble starch. 1 U of amyloglucosidase at pH 4.5 and 40 �Cwas defined as the

amount of enzyme required to release one micromole of D-glucose per minute

from soluble starch.

The bottle was capped and placed in a water bath at 37 �C with a

continuous (170 rpm) stirring via a stir bar. Fourteen bottles were incu-

bated for each sample, and two bottles were taken out after 1, 2, 4, 6, 8, 16

and 24 hours. After cooling to 25 �C, the pH of the mixture was decreased

to 2.5 to inactivate the enzyme activity with the addition 1.0M hydro-

chloric acid. The mixture was held at pH 2.5 for one hour before

increasing the pH to 6.0 via the addition of 1.0M sodium hydroxide.

Subsequently, the mixture was centrifuged (1500� g, 10min), supernatant

decanted, pellet washed (two times) with water and centrifuged (1500� g,

10min), and the pellet was then oven-dried at 37 �C for 5 hours. The dried

pellet (undigested portion of the CL wheat starch) was gently ground using

a pestle and mortar and was stored at room temperature in an airtight

container.


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8.2.5 Thermal properties

The gelatinization temperatures and the enthalpy of digested CL wheat starch

were measured by differential scanning calorimetry (DSC) (TA Instruments

Q100, TA Instruments, New Castle, DE). The total solids content of samples

were 33.3% (w/w, dry basis). After hydration for one hour at 25 �C, 30mg of

well-stirred sample suspensions were weighed into 40ml aluminium pans and

hermetically sealed immediately to prevent moisture loss. Scans were per-

formed from 10–130 �C at a constant heating rate of 10 �C/min. A sealed

empty pan was used as a reference and the DSC was calibrated using indium.

The gelatinization enthalpy (DH) and transition temperatures, namely the

onset temperature (To), peak temperature (Tp) and conclusion temperature

(Tc), were determined on the basis of the first-run DSC heating curves.

The DH was evaluated.

8.2.6 Microscopic observation

CLwheat starch digested after 1, 2, 4, 6, 8, 16 and 24 hours of incubation were

placed on microscope slides and observed under an Olympus BX-51 micro-

scope (Olympus, Tokyo, Japan) with a 40� objective. CL wheat starch before

digestion was also observed under a microscope for comparison with the

assayed CL wheat starch.

8.2.7 Scanning electron microscope (SEM)

The samples were sprinkled lightly onto a carbon double-sided adhesive

tape on metal specimen stubs, which were then coated with gold-palladium

(60 : 40 ratio) under vacuum with a Desk II Sputter/Etch Unit (Denton

Vacuum, LLC, Moorestown, NJ). Micrographs of the samples were obtained

at 1000� and 5000� magnifications by SEM (S-3500N, Hitachi Science

Systems, Ltd, Japan) at an accelerating potential of 20 kV using an X-ray

Detector-Link Pentafet 7021 (Oxford Instruments Microanalysis Limited,

Bucks, England).

8.2.8 Statistical analysis

All data were statistically analyzed by analysis of variance (ANOVA) using

Statistical Analysis Software (SAS) (version 9.2, SAS Institute, Cary, NC),

and the values are expressed as means � standard deviations from two

replicates, unless stated otherwise.


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8.3 RESULTS AND DISCUSSION

8.3.1 Effects of a-amylase/amyloglucosidase digestion onP content and chemical forms of the phosphateesters on starch

The P content of CL wheat starch after different incubation times with

a-amylase/amyloglucosidase is reported in Table 8.2. The initial P content

of CL wheat starch (at 0 hours) was 0.36%. The P content of CL wheat starch

remained constant for the first two hours but started to increase at four hours.

The P content progressively increased with incubation time, until it reached

0.56% at 24 hours, indicating that the molecules with higher P density were

more resistant to digestion. The regions with no or low bound P were

selectively removed, thus elevating the concentration of P in CL wheat starch

residue.

The 31P NMR spectra of phosphate derivatives of CL wheat starch and

their digestive residues after various a-amylase/amyloglucosidase incuba-

tion periods are also tabulated in Table 8.2. The effect of digestive enzymes

Table 8.2 Phosphorus content and levels of different phosphate esters of cross-linkedwheat starch and their digestive residues after various a-amylase/amyloglucosidaseincubation periods.

Phosphorus content (% dry basis)in the form of:

MSMP2

Digestiontime (h)

Totalphosphorus(%)�

Cyclic-MSMP1

C3 C6 DSMP3

0 0.36�0.01d 0.000 0.078 0.054 0.2281 0.36�0.02d 0.000 0.078 0.052 0.2302 0.36�0.00d 0.000 0.080 0.052 0.2294 0.38�0.02cd 0.004 0.090 0.057 0.2296 0.40�0.01c 0.004 0.089 0.061 0.2458 0.41�0.00c 0.004 0.099 0.062 0.24616 0.47�0.01b 0.004 0.104 0.069 0.29224 0.56�0.00a 0.005 0.136 0.080 0.338

�Values within columns bearing the same small letter superscript/s do not significantly differ

at (p<0.05).1Cyclic-MSMP, cyclic-monostarch phosphate.2MSMP, monostarch monophosphate positioned at C-3 and C-6.3DSMP, distarch monophosphate.


CH08GR 07/29/2013 16:22:49 Page 152

on theCLwheat starch started after four hours of incubation, causing an increase

in the cyclic-monostarch monophosphate (cyclic-MSMP) and monostarch

monophosphate (MSMP). The distarch monophosphate (DSMP) of CL

wheat starch started to increase drastically after six hours of incubation. The

remaining periods of incubation showed a significant increase in MSMP

and DSMP, whereas cyclic-MSMP remained constant until the digestion

was terminated.

The results suggest that cyclic-MSMP and other phosphate esters

increased due to hydrolysis of starch molecules that may not be linked

to P molecules, leaving the remaining starch granules that were cross-

linked to P molecules. 31P NMR spectra of CL wheat starch after 16 hours

of digestion, as shown in Figure 8.1b , has signals of cyclic-MSMP, MSMP

positioned at C3 and C6, inorganic phosphate (Pi) and DSMP at d15.4 ppm, d 1–3 ppm, d 3.5 ppm and d 0 to –1 ppm, respectively. The

cyclic-MSMP had stronger peak detention as compared to the spectra

before digestion (Figure 8.1a), which was consistent with the quantified

cyclic-MSMP (Table 8.2).

8.3.2 Thermal properties

The gelatinization transition temperatures, consisting of To, Tp, and Tc as

well as DH of CL wheat starches at different times of a-amylase/amylo-

glucosidase incubation, are reported in Table 8.3. Gelatinization of all

starches took place between 72 �C (for To) to 94 �C (for Tc), and an

endothermic peak was observed in the DSC curves of all samples. The

data was slightly higher than that reported by Woo & Seib (2002), presum-

ably due to higher P content (0.36%) in the commercial CL wheat starch in

our study as opposed to the laboratory-prepared CL wheat starch (0.32%).

There was a slight increase in the Tp and Tc for digested CL wheat starch

after 16 and 24 hours incubation. The DH for CL wheat starch at all

incubation times was comparable to the DH before digestion, which was

inconsistent with potato starch (Jiang & Liu, 2002) and maize starch (Shresta

et al., 2012). An amylose-lipid complex peak was absent for CL wheat starch

at all incubation times. Similar values of the thermal parameters imply that

the remaining CL wheat starch residual after the a-amylase/amyloglucosi-

dase digestion was likely to be intact, with little or no effect on starch granule

crystallinity. Native wheat starch (Colonna et al., 1988), native barley starch

(Lauro et al., 1999) and native maize starch (Brewer et al., 2012) had been

reported to have decreased crystallinity after enzymatic digestion. The

current result showed that cross-linking of wheat starch aided in the retention

of starch crystallinity up to 24 hours amylolysis.


CH08GR 07/29/2013 16:22:49 Page 153

8.3.3 Starch granular morphology before and afterenzyme digestion

Figures 8.2 and 8.3 represent light microscopic and SEM micrographs

respectively of CL wheat starch after various a-amylase/amyloglucosidase

incubation periods. The SEM results were correlated with the microscopic

C6

Pi

MSDP

(a) C3

15 10 5 0 –5 –10 –15 –20

Cylic-MSMP

(b)

Chemical shift (ppm)

Figure 8.1 31P nuclear magnetic resonance spectra of dextrins prepared from cross-linked wheat starch before (a) and after 16 hours of a-amylase/amyloglucosidasedigestion (b). Cyclic-MSMP: cyclic-monostarch monophosphate; C-3: monostarchmonophosphate positioned at C3; C-6: monostarch monophosphate positioned atC6; MSDP: monostarch diphosphate; Pi: inorganic phosphate.


CH08GR 07/29/2013 16:22:50 Page 154

evidence. However, the SEM micrographs provide a better observation of the

morphological changes of CLwheat starch granules after an incubation period

ranging from 0 to 24 hours.At the start (0 hours), the CL wheat starch granules had a smooth surface,

with round, shallow indentations of flower petal-like patterns which are

restricted to only some of the granules. In the first hour of digestion, mild

erosion on the surface of most starch granules was observed, indicating the

susceptibility of CL starch granules early during the incubation.

The progression of starch granule corrosion continued to be observed after

incubation from 1–4 hours. Scattered erosion on the surface of starch granules

increased and deepened with time. Most starch granules still, however,

remained intact, with the retention of the Maltese Cross pattern. After

incubation for six hours and eight hours, surface erosion of CL wheat starch

granules intensified, causing severe damage and the disappearance of granular

identity of some starch granules. The severity of damage on CL wheat starch

granules was more prominent after incubation for 16 hours and 24 hours, when

smaller fragments of broken starch granules were evident. However, some

starch granules remained intact and retained crystallinity, as depicted by the

Maltese Cross. This observation indicated that hydrolysis of CL wheat starch

granules by an a-amylase/amyloglucosidase cocktail was progressing con-

tinuously and did not stop until the 24 hours incubation was terminated.

CL wheat starch showed unequal granule degradation by digestive

enzymes at all incubation times. From 1–24 hours incubation, some of the

starch granules remained intact, while others had mild to extensive

Table 8.3 Thermal properties of cross-linked wheat starch and their digestive residuesafter various a-amylase/amyloglucosidase incubation periods.

Temperature (�C)Digestiontime (h) To Tp Tc DH (J/g)

0 72.0�0.2a 76.2�0.2c 89.8�0.7bc 10.7�0.3bc

1 72.2�0.1a 76.1�0.1c 90.4�0.2c 10.8�0.2bc

2 72.2�0.1a 76.1�0.1c 90.7�0.3c 11.8�0.1b

4 73.2�0.4a 77.3�0.5b 89.3�0.6bc 11.5�0.1b

6 72.3�0.3a 76.6�0.3c 91.5�0.5b 12.2�0.1a

8 72.3�0.1a 77.6�0.6b 91.7�0.5b 12.2�0.3a

16 73.2�0.1a 78.3�0.3ab 93.6�0.4a 11.3�0.4b

24 73.7�0.1a 78.9�0.0ab 93.8�0.3a 11.6�0.4b

Values within columns bearing the same small letter superscript(s) do not significantly differ at

(p>0.05).


CH08GR 07/29/2013 16:22:50 Page 155

Figure 8.2 Microscopic photos (40� objective) of cross-linked wheat starch atdifferent incubation times of a-amylase/amyloglucosidase digestion.


CH08GR 07/29/2013 16:22:53 Page 156

Figure 8.2 (Continued)


CH08GR 07/29/2013 16:23:2 Page 157

Figure 8.3 Scanning electron micrographs (1000� and 3000� magnifications) ofcross-linked wheat starch at different times of a-amylase/amyloglucosidasedigestion.


CH08GR 07/29/2013 16:23:8 Page 158

Figure 8.3 (Continued)


CH08GR 07/29/2013 16:23:11 Page 159

degradation. The same phenomenon was observed on the native wheat starch

granules (Figure 8.4 ), although the starch damage was more prominent at one

and two hours of digestion. This could be attributed to heterogeneous action of

the digestive enzymes on the wheat starch granules. Starch granules are also

Figure 8.4 Scanning electron micrographs (1000� and 3000� magnifications) ofnative wheat starch at one hour and two hours of a-amylase/amyloglucosidasedigestion.


CH08GR 07/29/2013 16:23:13 Page 160

described as not equally susceptible to enzymatic hydrolysis, greatly depend-

ing on the adsorption manner of amylases on the granule and the method of

isolation of the starch (Colonna et al., 1988; Oates, 1997).

Colonna et al. (1988) had a similar observation of unequal amylase

digestion on native wheat starch, in which some granules had minimal pitting

and a fewwere severely cratered. However, all large granules were completely

degraded.

The mechanism of enzyme attack on starch granules is due to exo-

corrosion (Shresta et al., 2012), endo-corrosion or a combination of both

(Manelius et al., 1997; Apinan et al., 2007). The microscopic and SEM results

suggest that the starch hydrolysis by amylase progressed, starting from the

surface and proceeding inside of native wheat and CL wheat starch granules

by exo-corrosion. In addition, the starch granules were most probably digested

by the digestive enzymes using the side-by-side mechanism, as observed by

Zhang et al. (2006) and Shresta et al. (2012), in which the enzyme digested on

the amylose and amylopectin as well as the amorphous and crystalline regions

(Zhang et al., 2006).

Unlike digested maize starches, which are reported to have holes or tunnels

deepening into the interior of granules, or ‘Swiss cheese’ shell appearance

(Robyt, 2009; Lauren et al., 2012), CL wheat starch granules (Figure 8.3) and

native wheat starch (Figure 8.4) had a roughened superficial surface and

exposure of layered internal structures that intensified with increased incuba-

tion time, causing the formation of cavities on some starch granules. In

addition, CL wheat starch was theoretically obstructed by phosphate groups

within the granule surface pores and channels, inhibiting the diffusion of

amylase molecules (Thompson et al., 2011).

8.3.4 Digestibility

The extent of digestibility of CL wheat starch at different incubation times (up

to 24 hours) was determined using the AOAC Method 2009.01. Wheat starch

was also assayed using the same method as control, since wheat starch is

easily hydrolyzed by digestive enzymes (Bj€orck et al., 1986). The amount of

digested starch after each incubation period is shown in Figure 8.5. Both

starches showed rapid digestion at one hour, and the rate of digestion slowed

until the end of the incubation period. The latter observation is in agreement

with that of Bertoft & Manelius (1992), which showed two stages of starch

digestion, the first being the initial rapid hydrolysis, followed by a slower and

more constant rate of hydrolysis.

For wheat starch, the amount of starch digested was close to 70% at one

hour of incubation and 99% at six hours, indicating a high susceptibility of


CH08GR 07/29/2013 16:23:13 Page 161

wheat starch granules to hydrolysis. The SEM results (Figure 8.4) showed the

severity of starch hydrolysis at the early stage of digestion (one hour and two

hours). Our result is different from that reported by Colonna et al. (1988), who

observed 74% and 91% starch hydrolysis after incubation for ten hours and 21

hours, respectively. The difference could be due to enzyme source. Colonna

et al. (1988) used Bacillus subtilus a-amylase (17.5 Phadebas Unit/mgprotein), whereas the current study used a combination of pancreatic a-amy-

lase (50U/ml) and amyloglucosidase (3.4U/ml). Pancreatic a-amylase had

been suggested to be the most effective enzyme to digest native starch,

followed by barley, bacterial and fungal amylases (Kimura & Robyt, 1995).

When subjected to a cocktail of a-amylase and amyloglucosidase at 37 �Cfor 24 hours, CL wheat starch was digested at a slower rate than native wheat

starch and, by 24 hours, 84% of the CL starch was digested. The cross-linking

treatment decreased the susceptibility of starch granules to digestive enzymes

due to the stabilizing effect of granules with a high degree of cross-linking

(Seib & Woo, 1999; Woo et al., 2009), therefore causing a slower rate of

digestion. The amount of digested CL wheat starch was negatively correlated

with P content, but had little effect on starch crystallinity.

Digestion time (hours)

Native wheat starch CL wheat starch

Dig

este

d st

arch

(%

)

0 5 10 15 20 25

100

90

80

70

60

50

40

30

20

10

0

Figure 8.5 Digested starch of cross-linked (CL) wheat starch and native wheat starchat different incubation times of a-amylase/amyloglucosidase digestion.


CH08GR 07/29/2013 16:23:13 Page 162

O’Brien&Wang (2009) proposed that amylopectin couldbemore reactive in

the cross-linking process, due to higher retained phosphate salts in the branched

structure of the polymer. Since amylopectin is comprisedof amorphous (tightly-

spaced branches) and crystalline regions (parallel glucans) (Oostergetel & van

Bruggen, 1989), phosphate groups may stabilize both amorphous and crystal-

line regions by promoting stronger interactions between the two regions.

Therefore, the undigested portion of the residual starch had increased stability

and was less susceptible to digestive enzymes, as depicted by the intact,

yet highly roughened starch granule surfaces observed in SEM micrographs

(Figure 8.3). Increased P content with increased incubation time showed that

phosphate groups were retained in the undigested residuals, reflecting the

incapability of digestive enzyme to cleave bonds close to a glucose linked

to a phosphate group.

Previous RS quantitation of CLwheat starch, using the EnglystMethod and

AOAC Method 991.43, showed 83% and 76%, respectively (Woo & Seib,

2002; Yeo & Seib, 2009; Thompson et al., 2011). While the Englyst Method

(incubation at 37 �C) and AOAC Method 991.43 (incubation at 100 �C) use120 minutes and 35 minutes of incubation time, respectively, AOAC Method

2009.01 incubates samples for 16 hours at 37 �C, yielding 25% RS content for

CL wheat starch.

The significantly lower RS content of CL wheat starch, as determined by

AOAC Method 2009.01, compared to the values obtained by the Englyst

Method and AOAC Method 991.43, raised questions about the validity of

AOAC Method 2009.01 to measure accurately RS content that is consistent

with in vivo human conditions. At 24 hours, RS content of CL wheat starch

further decreased to 16%, indicating that the digestive action of the

a-amylase and amyloglucosidase cocktail on CL wheat starch was still

progressing.

In humans, the transit time of food in the intestine takes 3–4 hours (Perera

et al., 2010). Long-term hydrolysis may lead to an increase in substrate

surface availability caused by amylase attack (Colonna et al., 1988). At 16

hours incubation time in AOAC Method 2009.01, a much lower yield of RS

content resulted, as indicated in the current study, and may not reflect in vivo

response, which is important in the food industry.

8.4 CONCLUSIONS

CL wheat starch was assayed using the digestive enzymes and incubation

conditions of AOAC Method 2009.01, and samples were collected at

predetermined times. The digestion of CL wheat starch continued to


CH08GR 07/29/2013 16:23:13 Page 163

increase up to 24 hours. The RS value obtained at 16 hours was lower than

the RS content measured by the Englyst Method and AOACMethod 991.43.

Microscopic and SEM results of the indigestible residues collected at the

predetermined times showed progressive degradation of CL wheat starch

granules. However, some starch granules remained intact, which explained

the comparable DSC data for CL wheat starch at all incubation times. The

mechanism of enzyme attack on the CL wheat starch was exo-corrosive, as

identified by the surface erosions and the non-existence of pinholes on the

granules.

Future studies will include assaying CL starches from different botanical

sources in order to understand further the mechanism of enzyme attack as

affected by cross-linking.

8.5 ACKNOWLEDGEMENTS

We thankMGP Ingredients, Inc. for donating the starch samples and Dr. Susan

Sun for the use of DSC. This is Contribution no. 13–233-J from the Kansas

Agricultural Experiment Station.

8.6 ABBREVIATIONS USED IN THIS CHAPTER

DH, gelatinization enthalpy; ANOVA, analysis of variance; CL, cross-linked;DSC, differential scanning calorimeter; P, phosphorus; RDS, rapidly digest-

ible starch; RS, resistant starch; SDS, slowly digestible starch; SEM, scanning

electron microscope; STMP, sodium trimetaphosphate; STPP, sodium tripo-

lyphosphate; Tc, conclusion temperature; To, onset temperature; Tp, peak

temperature.

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9 Biscuit Baking and Extruded SnackApplications of Type III ResistantStarch

Lynn Haynes, Jeanny Zimeri and Vijay AroraIngredient and Process Research, Mondelez International, USA

9.1 INTRODUCTION

Enzyme-resistant starch may be defined as the sum of starch and products of

starch degradation not absorbed in the small intestine of healthy individuals

(Eerlingen, 1994). It may be classified into four types, designated I to IV.

Physically inaccessible starch, locked in the plant cell, is classified as

type I. This type can be found in partially milled grains and seeds and legumes.

Native granular starch found in uncooked ready-to-eat starch-containing

foods, such as in bananas, is classified as type II resistant starch. Enzyme

susceptibility of type II resistant starch is reduced by the high density and

partial crystallinity of the granular starch.

The amount of type I and type II resistant starch is generally less than about

12% by weight, after grain milling and cooking, based on the amount of starch

contained in the starch source and in the formula. Type I and type II resistant

starches have low melting points and do not survive a baking process where

temperatures are substantially above 100�C and there is sufficient moisture in

the formula to gelatinize the starch (e.g. >20%). Type I and type II resistant

starch do not survive high shear processing such as extrusion, which would

disrupt granular integrity.

Starch may undergo a treatment process to obtain an indigestible starch

fraction. Depending upon the type of treatment, a type III resistant starch or a

type IV resistant starch may be produced. An indigestible starch fraction

which forms after certain heat-moisture treatments of the starch may be

present in, for example, cooked, cooled potatoes; this is type III enzyme-

resistant starch.

167




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In type IV resistant starch, the enzyme resistance is introduced by

chemically modifying or thermally modifying the starch. For example,

glycosidic bonds other than a-(1–4) and a-(1–6) bonds may be formed by

heat treatments. Other glycosidic bonds may reduce the availability of starch

for amylolytic enzymes. In addition, the digestibility of starch may be reduced

by chemical cross-linking (Woo & Seib, 2002). Production and use of such

ingredients may be subject to legal limitations imposed by the US Food and

Drugs Administration.

Details of a patented process and flour replacer performance in a cookie

formula have been published (Haynes et al., 2002), utilizing a process

comprised of gelatinization, nucleation/propagation and heat treatment stages

for an amylose extender corn starch (aeWx VII). The characteristics of a

thermal-stable, shear-stable enzyme-resistant starch type III RS (X150) and

its use in biscuit baking and extrusion applications, without the formation

of glycosidic bonds other than a-(1–4) and a-(1–6) glycosidic bonds,

are described.

9.2 THERMAL CHARACTERISTICS OF HEAT-SHEARSTABLE RESISTANT STARCH TYPE III INGREDIENT

The starch-based ingredient is comprised of about 60% by weight enzyme-

resistant starch type III, which has a melting point of about 150�C andwhich is

formed by gelatinization, followed by at least one cycle of crystal nucleation

and crystal growth or propagation. In the critical cooling step, the gelatinized

starch is cooled to a crystal nucleating temperature of >60�C, above the

melting point of amylopectin starch such that the nucleation of amylolipid

crystalline complex is not favoured. A crystal propagating temperature of

about 130�C is used to maximize the formation of the heat-stable RS III,

which has a thermal melt temperature of 150�C. A debranching enzyme, such

as pullulanase may be used to increase the yield of the high-melting enzyme-

resistant starch type III.

Shown in Figures 9.1 and 9.2 are the MDSC curves for enzyme-resistant

starch type III ingredient and isolate. In the MDSC technique, the material

being analyzed is heated at a steady rate with a programmed saw-tooth pattern

of heating and cooling imposed upon the steady rate. The amplitude of the

fluctuation in temperature allows a more precise analysis of the equilibrium

melting point because it separates overlapping thermal events such as

irreversible decomposition. In Figure 9.1, a single crystalline melt is observed

at about 150�C for resistant starch type III bulk ingredient. Enzyme-resistant


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Figure 9.1 Modulated differential scanning calorimetry (MDSC) curve for anenzyme-resistant starch type III ingredient (X150) or bulking agent, obtained froma single nucleation temperature of about 70�C and a propagation temperature ofabout 130�C.

Figure 9.2 MDSC curve for the isolated, heat-treated enzyme-resistant starch type IIIbulking agent.

Biscuit Baking and Extruded Snack Applications of Type III Resistant Starch 169

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starch can be isolated from the bulk ingredient by a method adopted and

modified from the Prosky method for Total Dietary Fibre in baked goods set

forth in AOAC Method 991.43.Shown in Figure 9.2 is the MDSC thermal profile of enzyme-resistant type

III starch isolated from the bulk ingredient.

The second stage nucleation/propagation temperature cycling is followed

by a third stage involving heat treatment of the enzyme-resistant starch type III

product. The heat treatment is conducted at a temperature of about 130�C for

about one hour at a moisture content of 18%. Figure 9.3 shows a MDSC curve

for the isolated, heat-treated enzyme-resistant starch type III bulking agent.

The heat treatment increases the total dietary fibre content and improves

baking functionality. Corresponding MDSC curves for type II resistant starch

isolate (Figure 9.4) and type II resistant starch bulk ingredient after heat

treatment (Figure 9.5) show no high temperature melting.An enzyme-resistant starch type III bulking agent produced from gelatini-

zation and recrystallization at high temperatures, followed by heat treatment,

has about 60% total dietary fibre and is resistant to enzymes such as

a-amylase, b-amylase, amyloglucosidase and pancreatin, and it provides a

reduced calorie or low calorie, highly functional ingredient for replacing flour

in baking or extruded applications. As a result of the very highmelting point of

Figure 9.3 MDSC curve for an isolated enzyme-resistant starch type III ingredient(X150) obtained from the bulk.


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Figure 9.4 MDSC curve for isolated, heat-treated enzyme-resistant granular starchtype II obtained by heat-treating granular type II (Novelose 240) bulking agent at130�C for one hour, followed by isolation of the RS from the heat-treated bulkingagent. (Novelose 240).

Figure 9.5 MDSC curve for heat-treated enzyme-resistant granular starch type II,obtained by heat treating an enzyme-resistant granular starch type II (Novelose 240)bulking agent.


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150�C, the enzyme-resistant starch ingredient is extremely heat-stable. The

resistant starch content of the type III resistant starch bulking ingredient

survives the high shear conditions of extrusion, enabling its use as a low

calorie, high fibre flour replacer in baking and extrusion applications.

9.3 APPLICATION TO BISCUIT BAKING: COOKIES

Cookies are baked products which generally contain three major ingredients,

i.e. flour, sugar and fat. Cookie quality attributes are their size and tender bite

(Miller & Hoseney, 1997). Also, cookies baked from soft wheat flour display

not only a large spread but also a uniform surface cracking pattern (Miller

et al., 1997). In cookie production, mixing disperses ingredients evenly and

promotes water absorption, rather than developing a true dough structure

(Huebner et al., 1999). Due to the high levels of fat and sugar, the development

of the gluten network is limited (Slade & Levine, 1994). Sugar is pre-

dissolved in water at a concentration in the formula water which exceeds

35% weight per weight, usually in the presence of formula fat. The dough

moisture is typically about 17–18%. The total solvent, sugar plus formula

water is about 64% (wt per cwt flour).

The cookie baking method used to evaluate resistant starch ingredient

performance is a standard wire-cut cookie baking method (AACC 10–53)

designed at the Nabisco Biscuit Company for the evaluation of ingredient

functionality and predictive correlation between sensory and mechanical

texture analysis (mechanical texture by Instron three-point bend or puncture

test). The AACC 10–53 was adopted as the approved method of the American

Association of Cereal Chemists after collaborative testing in 1992. Shown in

Table 9.1 is the formula used in the AACC 10–53 wire-cut cookie baking

method.

Cookie geometry and moisture bake-out is critical in the industrial setting,

which demands high through-put and automated packing of the product.

Correct cookie baking results in proper colour, taste and texture which, in turn,

influences consumer acceptance of the finished product. Achieving the right

geometry, colour and moisture balance is highly dependent upon ingredients

used in the formula.

In order to obtain a significant reduction in calories or an increase in total

dietary fibre per serving, usually 25% to 50% of the flour must be replaced. At

this level, solvent binding properties of flour replacers can disturb the colour

and moisture balance in the baked product. The water holding capacity of

conventional un-gelatinized wheat flour may be about 0.6 grams of water per

gram of dry flour. The bulking agent solvent holding properties ideally would


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approximate that of the flour that is being replaced. Typically, gelatinized

starch used as a baking ingredient has an extremely high water holding

capacity of about 10 g of water per gram of starch (Pomeranz, 1964). The

resistant starch type III ingredient bulking agent, ground to a particle size

distribution the same as that of conventional wheat flour, has a water holding

capacity of less than 3 g of water per gram of dry matter. The lower water

holding capacity generally results in lower viscosity dough and has a

beneficial effect upon spread and baking characteristics. The water holding

is substantially unaltered by baking.

Baking performance of the resistant starch ingredients is tested by replac-

ing 50% by weight of the wheat flour in the standard dough formulation with

enzyme-resistant starch ingredient to obtain a blend. For each of the resistant

starch compositions, the amount of wheat flour replacement, the ingredient

type, the enzyme-resistant starch content or yield, the AOAC total dietary fibre

content, the cookie width and the MDSC enthalpy are set forth in Table 9.2.

Also in Table 9.2 are the L�a�b� colour measurements.

As shown in Table 9.2 and in Figure 9.6, the heat-treated RS Type III

(X150) ingredient exhibits a superior baking characteristic of greater cookie

spread. Along with cookie geometry, the product colour and moisture are

closest to control in the cookie made with RS type III and are likely to deliver

optimum commercial production efficiencies.

The enzyme resistance and low caloric value of the very high melting

enzyme-resistant type III starch ingredient component is substantially

unaltered by baking. The pure (or 100% by weight) enzyme-resistant starch

Table 9.1 AACC 10–53 wire-cut cookie formula.

Ingredient Weight (g)Percwt

Ingredientmoist

Ingredientdry weight(g)

Low trans shortening 90 40.00 0.20% 89.82Sucrose, fine gran. 94.5 42.00 0.50% 94.03Salt 2.81 1.25 0.50% 2.8Non-fat dry milk 2.25 1.00 4.00% 2.16High fructose corn syrup 3.38 1.50 29.00% 2.4Ammonium bicarbonate 1.13 0.50 99.50% 0.01Sodium bicarbonate 2.25 1.00 50.00% 1.13Flour 225 100.00 13.00% 195.75water 49.5 22.00 100.00% 0Total weight 470.82 Dry solids 388.08Total moist 17.57%


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type III has a caloric value of essentially zero, or less than about 0.5 calories/

gram even after baking. The caloric value for starch which is not resistant

starch is about 4 calories per gram. Thus, a resistant starch type III ingredient

or bulking agent with at least a 30% yield of RS type III will exhibit a caloric

value of less than about 2.8 calories/gram (0.70� 4 cal/gþ 0.30� 0

cal/g¼ 2.8 calories/gram). The resistant starch type III ingredient or bulking

agent exhibits excellent baking characteristics in terms of oven spread, edge

contour, oil release, surface cracking, odour, colour or browning, mouth-feel

and texture. It may be used alone or in combination with other fibres or whole

grain flour to produce a healthy, wholesome product.

9.4 CRACKER BAKING

Chemically leavened cracker dough formulas differ from cookie formulas in

two important aspects. First, the level of sugar in a cracker formula, measured

as wt sugar/wt sugarþ formula water, is lower at about 28%. Second, the total

solvent (sugarþwater) per cwt of flour is lower at about 48% (wt per cwt

flour), most of which is water. The cracker formula moisture is about 35% of

formula, significantly higher than cookie formulas. The cracker formula

conditions of low sugar and higher formula moisture allow for the develop-

ment of wheat flour gluten, such that a cohesive, elastic dough can be formed

Figure 9.6 AACC 10–53 wire cut cookies made with 50% replacement of flour withresistant starch ingredient.


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into a thin sheet from which cracker dough pieces are cut. Ingredients with

high water absorption are undesirable, because a tough, dry, less elastic dough

results. To compensate, more formula water is added, which can contribute to

longer bake time, requiring greater amounts of heat energy to remove the

excess moisture.

Type II granular resistant starch, Type III resistant starch (X150), made

from preferred process as described (gelatinization, nucleation/propagation

and heat treatment stages), and Type IV chemically modified resistant starch

(Fibersym1 70 or its old name Fiberstar 70) are evaluated in a cracker

formula. Table 9.3 shows the formula amounts for sugar, which calculates to

24% (weight sugar divided by weight of sugar plus formula water), total

solvent (sugarþ syrupþwater) per cwt of flour of about 48% and the total

formula moisture of about 35%. The cracker dough is sheeted and dough

pieces cut and weighed and baked.

As a result of the lower solvent holding capacity of the high melting point,

thermally stable type III resistant starch (X150), formula water requirements

are lower and superior baking characteristics are achieved. Although there is

sufficient moisture to gelatinize starch, due to the high melting point of Type

III RS made by the preferred process there is no loss of enzyme resistance

upon baking. Differences in dough sheet toughness and extensibility, com-

pared to control flour, were noted for all dough made with resistant starch.

Reducing the flour content of the formula reduces the amount of flour gluten

protein available for dough development. Figure 9.7 shows the sheeting

property of dough when flour is replaced at the 50% level.

Replacement of flour with resistant starch makes the dough less tough/

cohesive and less extensible. Using stronger flour with the resistant starch

does help to improve dough toughness, although there is still a notable

Table 9.3 Model formula for chemically leavened cracker baking test.

Ingredients Total weight Per cwt

Stage 1 Pre-dissolved sucrose 6.12 11.08Stage 1 Corn starch 3.1 5.61Stage 1 Salt 0.61 1.10Stage 1 Mono calcium phosphate 0.31 0.56Stage 1 Sodium bicarbonate 0.16 0.29Stage 2 Low trans oil 2.75 4.98Stage 2 Tap water (110�F) 18.87 34.17Stage 2 High fructose corn syrup 1.84 3.33Stage 3 Flour 55.23 100.00

Total weight 88.99 161.13Total %moist 0.3532


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decrease from control. Addition of vital wheat gluten shows increase in

toughness and extensibility of the dough sheet, as vital wheat gluten level

increases, toughness and extensibility also increase.

The finished product is baked for the same length of time and the moisture

measured by percentage weight loss. Resistant starch type II (Novelose 260)

requires more water to form dough, so current bake time results in out-of-

specification cracker moisture. Tougher dough tends to result in crackers with

higher moisture. Type III resistant starch (X150) at a 50% level demonstrates a

mechanical break force closest to control, as shown in Figure 9.8.

Figure 9.7 Force required to stretch dough and distance stretched before breakingare shown for resistant starch ingredient used to replace flour before and after doughresting. Note: additional water (19.5g) is added to the Novelose sample becausedough did not develop with formula amount.

Figure 9.8 Finished product modulus and stress force for cracker made withresistant starch replacing 50% of regular flour. Final moisture contents of finishedproducts are also shown.


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Retention of enzyme resistance during baking was confirmed by AOAC

991.43 fibre analysis of the finished cracker product. The results of fibre

analysis are shown in Table 9.4.

9.5 EXTRUDED CEREAL APPLICATION

Ready-to-eat (RTE) cereals are typically constituted by a flour blend (wheat

flour, ground cereal fines, barley malted flour), which constitutes approxi-

mately 96% by weight of the solids, 2% granulated sugar, 1.25% fine granular

salt and 0.5% of other minor ingredients. Enzyme-resistant starch type III

(X150) has been used to replace up to 50% of the flour blend in RTE cereals.

In the processing of RTE cereals, the solid ingredients are extruded using

a twin-screw extruder at a moisture content of approximately 9% moisture

(w.b.) under medium-shear screw profile, configured as shown in Figure 9.9.

The extrudate expands at the exposure to atmospheric pressure just outside of

the die, referred to as ‘flashing off’ of moisture.

The very-high-melting, high-shear-surviving enzyme-resistant starch is

substantially unaltered by extrusion, i.e. it remains substantially enzyme

resistant and exhibits a reduced calorie value of less than about

1.6–2.0 kcal/gram (60–50% by weight RS type III, having a melting point

or endothermic peak temperature of at least 140�C, as determined by MDSC),

as determined by fibre analysis. Enthalpy values for the high-melting enzyme-

resistant starch in the cereal are typically in the order of 6 Joules/g (based on

grams of resistant starch in the flour blend), at a temperature of from 130�C to

about 165�C. This is highly advantageous for producing reduced-calorie

extruded cereals because, if the crystal structure that provides enzyme-

resistance is destroyed or melts during extrusion, and if the crystal recrys-

tallizes into a lower-melting form which is not enzyme-resistant, then calorie

reduction will not be achieved in the extruded product. For example, when

Table 9.4 Results of AOAC method 991.43 fibre analysis in finished crackers.

ProductFlourreplacer

Productflour

Flourreplacement

% Dietaryfibre(theoretical)

% Dietaryfibre(actualanalysis)

% Fibreretentionafterprocessing

Cracker,

unoiled

Control 75.8% 0% 2.2 2.4 109%

X150 75.8% 50% 19 17.3 91%

Novelose

260

75.8% 50% 22.7 19.9 88%


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using a type II or type IV resistant starch, the calorie reduction would be less,

since these types of resistant starch do not survive high temperature or high

shear processing, such as extrusion.

The RS type III ingredient gives the extruded RTE cereal excellent

extrusion characteristics in terms of density, moisture content, texture (brit-

tleness) and cell structure.

9.5.1 Preparation of extruded RTE cereal and analysis

The extrusion functionality of an enzyme-resistant starch type III prepared in

accordance to the above procedure was compared to the extrusion function-

ality of a commercially available, enzyme-resistant starch type II ingredient

(Hi-MaizeTM 1043, produced by National Starch and Chemical Co.) and

a commercially available, enzyme-resistant starch type IV ingredient

(Fibersym1 70, produced by MGP Ingredients) using conventional, non-

heat-treated wheat flour as Control.

Hi-MaizeTM 1043 has the following characteristics, as claimed by National

Starch and Chemical Co.:

� Moisture: 13% maximum;� Total dietary fibre (AOAC method 991.43): 60% minimum (dry basis);� Calories: approx. 1.6 kcal/g.

Figure 9.9 Medium-shear screw profile used in extruder.


CH09GR 07/29/2013 16:34:11 Page 180

Fibersym1 70 (also known by its old name as Fiberstar 70) has the

following characteristics, as claimed by MGP:

� Moisture: 10.6% typical;� Total dietary fibre (AOAC method 991.43): 70% minimum (dry basis);� Calories: approx. 3.6 kcal/g (not corrected for insoluble fiber)

Extrusion functionality was evaluated bymeasurement of density, moisture

content, texture (brittleness) and cell structure. Resistant starch ingredients

that resulted in cereal properties as close to the properties achieved with the

wheat flour control were considered to have the best extrusion functionality.

Total dietary fibre was used to determine thermal and shear stability of the

resistant starch ingredients.

Control was produced following a standard formula for extruded, expanded

RTE cereals. In test samples, 50% of the total flour weight in the formula

(including wheat flour, ground cereal fines and barley malted flour) was

replaced by a resistant starch, namely RS III (X150) (variable 1), Hi-Maize

1043 (variable 2), or Fibersym 70 (variable 3), with the objective of testing

their functionality as flour replacers.

9.5.1.1 Formulas

The formulas are shown in Table 9.5.

9.5.1.2 Extrusion

The operating conditions used in the extruder to produce the cereal samples

are shown in Table 9.6.

9.5.1.3 Results

As shown in Table 9.7, the bulk density of the three test samples was lower

than that of the control, in the following descending order: Fibersym

70>RSIII>Hi-Maize 1043. Bulk density is an important parameter when

dealing with packing of a standard weight of cereals in a standard volume.

Also as shown in Table 9.7, resistant starch raw materials RSIII, Hi-Maize

1043 and Fibersym 70 had MDSC enthalpies of 8.39 J/g, 0 J/g and 0 J/g,

respectively, at temperatures above 140�C. Hi-Maize 1043 presented an

endotherm at 101.6�C and an enthalpy of 4.4 J/g, while Fibersym 70 presented

an endotherm at 74.9�C and an enthalpy of 11.71 J/g, representing low-

melting resistant starches.


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The control did not present a MDSC enthalpy above 140�C, indicating thatno enzyme-resistant starch was present and that none was created during

processing. Cereal samples containing RSIII presented a MDSC enthalpy of

9.05 J/g (based on g of RSIII in the flour blend), comparable to that of the

RSIII raw material, indicating that the RSIII ingredient was stable during

extrusion. In addition, it presented a high melting point of 151.2�C, corre-sponding to enzyme-resistant starch ingredients. Since it remained unchanged

during extrusion, RSIII was very identifiable in the final product.

Cereal samples containing Hi-Maize 1043 presented a MDSC enthalpy of

4.35 J/g (based on g of Hi-Maize 1043 in the flour blend) at 144.7�C, value,which was equivalent to the enthalpy exhibited by the Hi-Maize 1043

ingredient, but presented a shift in temperature of about 45�C with respect

to the original melting temperature of 101.6�C in the Hi-Maize 1043

ingredient. Thus, generation of crystalline resistant starch, as expected

from high temperature/moisture processes, occurred during extrusion and/

or DSC analysis. A smaller enthalpy than that for RSIII represented a sample

with less degree of crystallinity and, thus, less content of enzyme-resistant

starch. In addition, the generated enzyme-resistant starch had a lower melting

point than that of RSIII.

Cereal samples containing Fibersym 70 presented a MDSC enthalpy of

1.2 J/g (based on g of Fibersym in the flour blend) at 122�C, a value that was

Table 9.7 Final product property measurements.

Bulkdensity(g/cm3)

Moisture(%)

MDSC enthalpy@ >140�C(J/g)�

RS meltingpeaktemperature(�C) L�, a�, b�

RSIII, raw material – – 8.39 151.2 –

Hi-MaizeTM 1043, raw

material

– – 0 101.6 –

Fibersym�R 70, rawmaterial

– – 0 74.9 –

Control cereal 0.452 9.73 0 – 70.61, 3.81,

25.33RSIII cereal, 50% flour

replacement

0.309 9.99 9.05 151.2 68.49, 4.62,

25.59

Hi-MaizeTM 1043

cereal, 50% flour

replacement

0.195 10.02 4.35 144.7 75.29, 2.47,

22.26

Fibersym�R 70 cereal,

50% flour

replacement

0.365 7.81 0 122 73.39, 3.21,

26.74

� Joules per g of flour, g of RSIII in flour blend, g of Hi-MaizeTM in flour blend, or g of Fibersym�R inflour blend, respectively.


CH09GR 07/29/2013 16:34:16 Page 184

very different in both magnitude and temperature from the original Fibersym

70 raw material. Since Fibersym 70 is a cross-linked starch, as opposed to

granular and retrograded starches in the case of Hi-Maize 1043 and X-150,

enthalpy might not correlate well with the amount of enzyme-resistant starch.

A better measurement would correspond to amount of total dietary fibre, as

will be discussed later.

As shown in Table 9.8, the theoretical total dietary fibre content in the

resistant starches corresponded to: RSIII¼ 50%; Hi-Maize 1043¼ 62%;

Fibersym 70¼ 70% (although it assayed at 80% using the AOAC 991.43

method), based on fibre content of ingredient declared in specifications.

Although cereal samples containing Hi-Maize 1043 had a higher theoreti-

cal dietary fibre content than cereal samples containing RSIII, calculated

based on fibre content of the ingredient declared in the specifications, the

measured AOAC total dietary fibre for cereal samples containing RSIII was

higher than that for cereal samples containing Hi-Maize 1043. The calculated

% fibre retention (i.e. fibre that survived processing plus that being generated

during processing) corresponded then to 96% for RSIII cereals and only 67%

for Hi-Maize 1043 cereals.

Table 9.8 Fibre content of extruded, RTE cereals.

% totaldietaryfibre,theoreticala

% AOACtotal dietaryfibreb

% fibre inRS ingredientafterprocessingc

% fibreretentionin RSingredientd

RSIII ingredient 50 – – –Hi-MaizeTM ingredient 62 – – –Fibersym�R 70

ingredient70, min. 80 – –

Control cereal 5.58 7.7 – –RSIII cereal, 50% flour

replacement23.3 22.4 48 96

Hi-MaizeTM 1043cereal, 50% flourreplacement

28.9 19.4 42 67

Fibersym�R 70 cereal,50% flourreplacement

37.3 12.60 27 34

aCalculated based on fibre content of ingredient declared in specifications.bAs measured by AOAC 991.43.cCalculated based on measured AOAC TDF in final product.dCalculated based on % total dietary fibre in RS ingredient, declared in specifications, vs. % fibre in RS

ingredient after processing.


CH09GR 07/29/2013 16:34:16 Page 185

Although cereal samples containing Fibersym 70 had the highest theoreti-

cal dietary fibre content than the rest of the samples, calculated based on

measured fibre content of the ingredient (80%), the measured AOAC total

dietary fibre for cereal samples containing Fibersym 70 was the lowest of all

the samples. The calculated percentage fibre retention corresponded to only

34%, which suggests that Fibersym 70 did not completely survive high

temperature and high shear conditions present during extrusion.

Cereal brittleness is presented in Figure 9.10. A high peak force and low

peak distancewould correspond to a brittle sample, while a low peak force and

high peak distancewould correspond to a less brittle sample. The x- and y-axis

error bars represent �1 standard deviation, while the diamonds represent

averages. Overlapping error bars represent samples that were not significantly

different from one another. In this case, control, X-150 and Fibersym

70 samples did not present a significantly different peak force or a signifi-

cantly different peak distance, indicating that they possessed a similar

brittleness. Hi-Maize 1043 samples showed a larger peak distance than the

rest of the samples, indicating a lower brittleness. Hi-Maize 1043 samples did

not present a significantly different peak force than RSIII or Fibersym

70samples, but theywere significantly softer (lowerpeak force) than the control.

As shown in Figure 9.11, air cells were more numerous and larger in the

control than in the rest of the samples, a characteristic which can be correlated

to bulk density. Cell structure is the result of expansion during extrusion,

governed by moisture content, moisture flash-off and flour blend (matrix)

physicochemical properties.

As shown in Figure 9.12, cereal samples containing RSIII showed superi-

ority in bowl life when compared to the other two resistant starch-containing

Figure 9.10 Extruded cereal brittleness.


CH09GR 07/29/2013 16:34:16 Page 186

samples (i.e. RSIII samples force vs. distance (deformation) behaviour after

soaking in 8�C water for 30 minutes was closer to the control than that of

Fibersym 70 and Hi-Maize 1043). For example, if a distance of 10mm was

chosen on the force vs. distance plot, it can be seen that the control was harder

(higher force) than the rest of the samples, and that RSIII was harder (higher

force) than Hi-Maize 1043 and Fibersym 70. The same behaviour was

Figure 9.11 Cell structure of extruded cereals.

0 5 10 15 20 25 30 35

300000

250000

200000

150000

100000

50000

0

–50000

Force (g)

Distance (mm)

Control RS III

Hi-Maize™

Fibersym® 70

Figure 9.12 Extruded cereal bowl life.


CH09GR 07/29/2013 16:34:17 Page 187

observed at deformations above 23mm. Between 15–23mm of deformation,

Fibersym 70 presented a higher force than both Hi-Maize 1043 and RSIII, but

its force vs. deformation curve behaved in a very different manner than the

control.

In summary, and as shown by the results discussed above, samples

containing RSIII had a superior extruding functionality and dietary fibre

content compared to Hi-MaizeTM 1043 and Fibersym1 70.

The melting profile or thermal characteristics of the resistant starches Type

II, III and IV, as well as the cereals containing these ingredients, were

determined by modulating differential scanning calorimetry (MDSC). Results

of the MDSC analysis for Hi-Maize 1043, RS III (X150) and Fibersym 70 are

shown in Figure 9.13.

� For Hi-Maize 1043, the onset of melting occurs at about 93�C, the

endothermic peak or melting point is about 101�C and the endpoint of

melting occurs at about 112�C.

Figure 9.13 MDSC curves for Hi-MaizeTM 1043 (RSII), RSIII (X150) and Fibersym1

70 (RS IV) ingredients, and for cereals containing these raw ingredients. 1:1 dilutionin distilled water.


CH09GR 07/29/2013 16:34:19 Page 188

� For RSIII, the onset of melting occurs at about 135�C, the endothermic peak

or melting point is about 151.2�C and the endpoint of melting occurs at

about 165�C.� For Fibersym 70, the onset of melting occurs at about 68�C, the endother-

mic peak or melting point is about 75�C and the endpoint of melting occurs

at about 95�C.

The software calculates the enthalpy of the endothermic peak in J/g.

Results of the MDSC analysis for cereals containing resistant starch

ingredients are also shown in Figure 9.13.

� For Hi-Maize-containing cereal, the onset of melting occurs at about

135�C, the endothermic peak or melting point is about 145�C and the

endpoint of melting occurs at about 155�C.� For RSIII-containing cereal, the onset of melting occurs at about 135�C, the

endothermic peak or melting point is about 152�C and the endpoint of

melting occurs at about 165�C.� For Fibersym 70-containing cereal, the onset of melting occurs at about

107�C, the endothermic peak or melting point is about 122�C and the

endpoint of melting occurs at about 133�C.

Figure 9.14 MDSC curve for Novelose 330 (RSIII). 1:1 dilution in distilled water.


CH09GR 07/29/2013 16:34:20 Page 189

MDSC results for Novelose 330 (National Starch & Chemical Co.,

Bridgewater, NJ), another commercially available type III resistant starch,

are shown in Figure 9.14. The onset of melting occurs at about 104� C, theendothermic peak or melting point is about 118.2 and the endpoint of melting

occurs at about 135� C. Thus, even though this is a type III resistant starch, itmelts at a lower temperature than RSIII, indicating that it would not survive

high-temperature extrusion conditions such as those used in breakfast cereal

manufacture, having very little functionality as a fibre.

REFERENCE S

Anderson, S., Haynes, L., Zhou, N., Slade, L., Levine, H., Kweon, M., Locke, J., Zimeri, J.(2005). Reduced-Calorie Flour Containing Type 3 Resistant Starch Used In ModelCracker System. AACCI Annual Meeting, September 11, 2005, Orlando, FL.

Eerlingen, R. (1994). Formation, Structure and Properties of Enzyme Resistant Starch.Doctoral dissertation. Katholieke Universiteit Leuven. Available at: http://www.biw.kuleuven.be/lmt/labolmc/DOCS/Eerlingen.htm

Haynes, L, Gimmler, N., Locke, J.P.III, Kweon, M-R, Slade, L., Levine, H. Production ofan enzyme-resistant starch for a reduced calorie flour replacer. United States Patent,US6352733, March 5, 2002.

Huebner, F.R., Bietz, J.A., Nelsen, T., Bains, G.S., Finney, P.L. (1999). Soft Wheat Qualityas Related to Protein Composition. Cereal Chemistry 76(5), 650–655.

Kweon, M., Haynes, L., Locke, J., Slade, L., Levine, H. (2004). Baking functionality of areduced-calorie flour replacer containing type-3 resistant starch. AACC/TIA JointMeeting, Sep 19 – 22, 2004, San Diego, CA.

Miller, R.A., Hoseney, R.C. (1997). Factors in Hard Wheat Flour Responsible for ReducedCookie Spread. Cereal Chemistry 74(3), 330–336.

Miller, R.A., Hoseney, R.C., Morris, C.F. (1997). Effect of Formula Water Content on theSpread of Sugar-Snap Cookies. Cereal Chemistry 74(5), 669–671.

Pomeranz, Y. (1964). Wheat: Chemistry and Technology, Vol. II, 333. Washington StateUniversity: Pullman, WA.

Slade, L., Levine, H. (1994). Structure-function relationships of cookie and crackeringredients. In: Faridi, H. (ed). The Science of Cookie and Cracker Production,23–141. Chapman & Hall: New York.

Woo, K.S., Seib, P.A. (2002). Cross-Linked Resistant Starch: Preparation and Properties.Cereal Chemistry 79(6), 819–825.

Zimeri, J.E., Haynes, L., Olson, A., Arora, V.K., Slade, L., Levine, H., Kweon, M.Production of low calorie, extruded, expanded foods having a high fiber content.United States Patent, US7648723, January 19, 2010.


http://www.biw.kuleuven.be/lmt/labolmc/DOCS/Eerlingen.htm

http://www.biw.kuleuven.be/lmt/labolmc/DOCS/Eerlingen.htm

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10 Role of Carbohydrates in thePrevention of Type 2 Diabetes

Thomas M.S. WoleverDepartment of Nutritional Sciences, University of Toronto, Canada; Divisionof Endocrinology and Metabolism, St. Michael’s Hospital, Canada

10.1 INTRODUCTION

The main objectives of this chapter are to review basic facts about diabetes

mellitus (definition, types, prevalence and risk factors) and to understand how

dietary carbohydrates affect the risk of developing type 2 diabetes and some of

the mechanisms by which dietary carbohydrates may be able to prevent type

2 diabetes, namely reduced postprandial glucose responses and increased

colonic fermentation.

10.2 BACKGROUND

10.2.1 Definition of diabetes

Diabetes is a condition in which the body either cannot produce insulin, or

cannot use the insulin it produces, resulting in a rise in the blood glucose

concentration. Therefore, the diagnosis of diabetes is made based on the

finding of high blood glucose. For men and non-pregnant women, diabetes is

present if any one of the following three conditions is met (Canadian Diabetes

Association Clinical Practice Guidelines Expert Committee, 2008):

� fasting plasma glucose equal to or greater than 7.0mmol/l (126mg/dl);� random (i.e. at any time of the day) plasma glucose equal to or greater than

11.1mmol/l (200mg/dl); or� a plasma glucose equal to or greater than 11.1mmol/l (200mg/dl) two hours

after consuming 75 g glucose (oral glucose tolerance test).

191




3GCH10 07/20/2013 8:53:8 Page 192

10.2.2 Types of diabetes

There are three main types of diabetes: type 1, type 2 and gestational diabetes:

� Type 1 diabetes, formerly known as insulin-dependent diabetes or juvenile

onset diabetes, is an autoimmune disease in which the insulin-producing

cells of the pancreas are destroyed, so that the body no longer produces any

– or only a very small amount of –insulin. Type 1 diabetes usually develops

in children or adolescents, affects about 5–10% of people with diabetes and

is treated with lifelong injections of insulin.� Type 2 diabetes, formerly known as noninsulin-dependent diabetes or

maturity onset diabetes, occurs when the pancreas cannot produce enough

insulin to meet the body’s needs and/or the body is unable to respond

normally to the actions of insulin (insulin resistance). Type 2 diabetes

usually develops in middle-aged or elderly adults (although it can occur in

children), affects about 90–95% of people with diabetes and is treated by

diet and exercise and, if necessary, oral medications and/or insulin.� Gestational diabetes is high blood glucose that develops during pregnancy.

It affects about 2–4% of pregnant women and is treated by diet and, if

necessary, insulin. Blood glucose levels usually return to normal after

delivery.

10.2.3 Complications of diabetes

Diabetes has serious complications, including heart disease, kidney disease,

eye disease, impotence and nerve damage, which are caused by high blood

glucose. Treatment of diabetes to reduce blood glucose levels is known to

prevent, delay the onset of or reduce the severity of these complications.

10.2.4 Prevalence of diabetes

Diabetes is a major public health issue because of the increased morbidity and

mortality associated with it, the high cost of treatment, and the fact that its

prevalence is rising rapidly in all parts of the world. In 1998, it was estimated

that there were 135 million people in the world with diabetes and that this

number would more than double to 300 million by 2025 (King et al., 1998).

However, the prevalence of diabetes is increasing faster than previously

expected; it is now estimated that there are 285 million people worldwide

who are affected by diabetes, and that this number is increasing by seven

million per year to reach 390 million by 2025 (Canadian Diabetes Associa-

tion, 2010).


3GCH10 07/20/2013 8:53:8 Page 193

10.2.5 Risk factors for type 2 diabetes

Since most people with diabetes have type 2 diabetes and since diet plays a

larger role in the prevention of type 2 than type 1 diabetes, the rest of this

chapter is concerned with type 2 diabetes. Consideration of dietary strategies

to prevent type 2 diabetes is particularly important for people at high risk of

developing the condition. Major factors associated with increased risk for type

2 diabetes include (Canadian Diabetes Association Clinical Practice Guide-

lines Expert Committee, 2008):

� being overweight or obese (body mass index over 25 kg/m2);� age over 40 years; family history of diabetes in a first degree relative (parent

or sibling);� being a member of a high risk population (Aboriginal, African, Hispanic,

Asian or South Asian);� having previously had gestational diabetes or high blood glucose (impaired

fasting glucose which is fasting glucose between 5.6–6.9mmol/l or

100–125mg/dl or impaired glucose tolerance, which is blood glucose

two hours after oral glucose tolerance test between 7.8–11.0mmol/l or

140–199mg/dl);� having high serum triglycerides or low serum high-density lipoprotein

(HDL) cholesterol;� the presence or history of peripheral-, coronary- or cerebro-vascular

disease;� the presence or history of schizophrenia.

10.3 CARBOHYDRATES AND RISK OF TYPE 2 DIABETES

With respect to the prevention of type 2 diabetes, there is strong evidence that

the quality of dietary carbohydrate is more important than the quantity. There

is little evidence that the amount of carbohydrate consumed influences risk for

type 2 diabetes. In six out of seven recent prospective studies in which it was

examined, the amount of carbohydrate consumed had no significant effect on

type 2 diabetes (see Table 10.1).

10.3.1 Markers of carbohydrate quality

The quality of dietary carbohydrate has traditionally been assessed by

considering simple versus complex carbohydrates. However, there is no

evidence that a high intake of simple carbohydrates causes diabetes (Janket

Role of Carbohydrates in the Prevention of Type 2 Diabetes 193

3GCH10 07/20/2013 8:53:9 Page 194

et al., 2003; Hodge et al., 2004), although consumption of sugar-sweetened

beverages may increase diabetes risk (Schulze et al., 2004a).

Measures of carbohydrate quality which do influence risk for type

2 diabetes include cereal fibre and glycemic index (Table 10.1). Cereal fibre

is considered here to be a qualitative indicator of carbohydrate intake,

because it represents a small proportion of total carbohydrate (usually less

than 10%) and is a marker of the types of carbohydrates consumed. In six out

of seven studies in which it was examined, a high intake of cereal fibre was

associated with a significant reduction in the relative risk of developing type 2

diabetes; the mean reduction in risk was about 30%.

The other marker of carbohydrate quality is glycemic index (GI). GI is

defined as 100� F/R, where F is the incremental area under the glycemic

response curve (AUC) elicited by a portion of food containing 50 g available

carbohydrate, and R is the mean AUC elicited by 50 g available carbohydrate

from the reference food or glucose tested 2–3 times by the same subject. The

GI is the mean of these values for a group of subjects (typically n¼ 10)

(Brouns et al., 2003; Wolever, 2006). In seven out of eight studies in which it

was examined, a high GI diet significantly increased risk for type 2 diabetes,

with the mean increase in risk being about 30%.

A marker of both carbohydrate quality and quantity is glycemic load,

defined as GI� g, where GI is the food glycemic index and g is the amount of

available carbohydrate in the food. Glycemic load is considered by many to be

Table 10.1 Relative risk of type 2 diabetes for 5th vs. 1st quintiles of intake of totalcarbohydrate, cereal fibre, glycemic index and glycemic load.

Totalcarbohydrate

Cerealfibre

Glycemicindex

Glycemicload

Salmeron et al.,1997a

ns 0.70 1.37 ns

Salmeron et al.,1997b

ns 0.72 1.37 1.47

Meyer et al., 2000 ns 0.64 ns nsSchulze et al.,

2004bns 0.63 1.59 ns

Hodge et al., 2004 ns ns 1.23 nsZhang et al., 2006 ns 0.77 1.30 1.61Villegas et al., 2007 1.28 – 1.21 1.34Krishnan et al.,

2007– 0.82 1.23 1.22

Values are relative risks which are significantly different from 1 for fully adjusted models; ns¼ non-

significant effect; ‘–’ is shown for studies where no result is given.


3GCH10 07/20/2013 8:53:9 Page 195

a more complete indicator of the physiological impact of dietary carbohy-

drate. However, it is not a consistent predictor of diabetes risk, with a

significant effect only found in four out of the eight studies in which it

was examined (Table 10.1).

10.4 PATHOGENESIS OF TYPE 2 DIABETES

Prospective studies suggest that the quality of dietary carbohydrate (high

cereal fibre intake and/or low GI) is more important than the quantity

consumed for preventing type 2 diabetes. To determine if there are plausible

mechanisms by which high cereal fibre and low GI diets reduce the risk for

type 2 diabetes, it is necessary to understand the pathophysiological mecha-

nisms which cause type 2 diabetes.

Many factors influence blood glucose, but perhaps the most important is

insulin. Insulin controls the blood glucose concentration via a classical

feedback loop. A rise in blood glucose stimulates the secretion of insulin

from the beta-cells of the pancreas, and the resulting rise in blood insulin

stimulates muscle and adipose tissue to increase glucose uptake, hence

causing blood glucose to fall. The ability of a rise in plasma glucose to

stimulate insulin secretion is termed insulin secretion, and the ability of

insulin to stimulate glucose uptake from the blood is termed insulin sensitivity.

In normal subjects, there is a hyperbolic relationship between insulin secretion

and insulin sensitivity (Clausen et al., 1996). Thus, people with high insulin

sensitivity (or low insulin resistance) have low insulin secretion in response to

a rise in blood glucose, while those with low insulin sensitivity (or high insulin

resistance) have high insulin secretion. The product of insulin secretion and

insulin sensitivity is termed disposition index, which is a marker of beta-cell

function.

It is commonly thought that diabetes develops because of decreased insulin

sensitivity (or increased insulin resistance). However, the relationship

between insulin secretion and insulin sensitivity suggests that blood glucose

remains normal as long as changes in insulin sensitivity are compensated for

by changes in insulin secretion. Type 2 diabetes develops when the disposition

index falls – that is, when insulin secretion cannot be increased enough to

compensate for reductions in insulin sensitivity (Kahn, 2003).

Most studies investigating the role of diet on the pathogenesis of type

2 diabetes only measure insulin sensitivity, but this does not necessarily

indicate risk for type 2 diabetes, because the deleterious effects of reduced

insulin sensitivity or the beneficial effects of increased insulin sensitivity on


3GCH10 07/20/2013 8:53:9 Page 196

blood glucose could be compensated for by compensatory changes in insulin

secretion. Indeed, studies in which both insulin secretion and insulin sensi-

tivity have been measured have shown that factors which increase insulin

sensitivity, such as exercise (Kahn et al., 1990), are accompanied, at least in

the short term, by a compensatory reduction in insulin secretion. Conversely,

factors which cause a reduction in insulin sensitivity, such as pregnancy

(Catalano et al., 1993) or use of nicotinic acid (Kahn et al., 1989) are

associated with compensatory increases in insulin secretion, such that the

disposition index did not change.

The dynamic relationship between insulin secretion and insulin sensitivity

makes teleological sense, because it allows blood glucose control to be

maintained despite changes in insulin sensitivity that normally occur through-

out life due to physiological factors such as puberty, pregnancy and changes in

body composition that occur during aging, and also by environmental factors

such as short-term changes in activity and the quality and quantity of food

intake.

The relative importance of changes in insulin secretion and changes in

insulin sensitivity in causing the development of type 2 diabetes has been

shown in two studies in which both were measured longitudinally in the same

subjects (Weyer et al., 1999; Festa et al., 2006). These studies show that a

reduction in insulin sensitivity is not necessarily associated with the develop-

ment of diabetes but, rather, it is the change in insulin secretion that

determines whether an individual develops diabetes or not. In the larger

study of this kind, oral glucose tolerance was assessed, and insulin secretion

and insulin sensitivity were measured, using a frequently sampled intravenous

glucose tolerance test in nearly 800 subjects at baseline and after a mean

period of 5.2 years follow-up.

Figures 10.1a and 10.1c show the results for subjects who had normal

glucose tolerance at baseline divided into three groups: those who remained

normal at follow-up; those who developed impaired glucose tolerance

(IGT); and those who developed diabetes. All three of these groups of

subjects gained weight and became more insulin resistant (less insulin

sensitive) with time. What distinguished them was that subjects who

remained normal increased insulin secretion more than those who developed

impaired glucose tolerance, while those who developed diabetes had no

change in insulin secretion. Similar results are seen for subjects who had

impaired glucose tolerance at baseline (Figures 10.1b and 10.1d). Remark-

ably, some subjects with impaired glucose tolerance at baseline became

normal at follow-up, despite the fact that they became more insulin resistant;

the increase in insulin secretion was large enough to overcome the reduction

in insulin sensitivity.


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10.5 EFFECT OF ALTERING SOURCE OR AMOUNT OFDIETARY CARBOHYDRATE ON INSULINSENSITIVITY, INSULIN SECRETION ANDDISPOSITION INDEX

The glycemic impact of the diet can be reduced by changing the source of

dietary carbohydrate (i.e. using low-GI foods) or by reducing the amount of

(a)NormalIGTDiabetes

(c)

(b) (d)

Normal at baseline IGT at baseline

Change over 5.2 years Change over 5.2 years

Weight Si AIR Weight Si AIR

Insu

lin s

ecre

tion

3

2

1

0

–1

0 1 2 3 0 1 2 3

100

80

60

40

20

0

Insulin sensitivity Insulin sensitivity

IGT → N (n = 66)

IGT → IGT (n = 95)

IGT → D (n = 59)

N → N(n = 350)

N → IGT(n = 105)

N → D(n = 40)

N → NN → IGTN → D

IGT → NIGT → IGTIGT → D

Figure 10.1 Changes in body weight, insulin sensitivity (Si) and insulin secretion (AIR,area under the insulin response curve after intravenous glucose) over 5.2 years insubjects who had normal glucose tolerance at baseline (a and c) and subjects whohad impaired glucose tolerance at baseline (b and d), and whose glucose toleranceat follow-up had remained, or become, normal (N), impaired (IGT) or diabetic (D).Adapted from Festa et al. (2006).


3GCH10 07/20/2013 8:53:9 Page 198

carbohydrate consumed. Since epidemiological studies suggest that carbohy-

drate quality is associated with risk for diabetes, but that carbohydrate

quantity is not (Table 10.1), we hypothesized that, on weight-maintaining

diets, reducing diet GI without reducing carbohydrate intake would have a

more beneficial effect on insulin sensitivity and insulin secretion than a

moderate reduction in carbohydrate intake.

To test this, we studied 35 subjects with IGT, whom we identified by

screening subjects with risk factors for type 2 diabetes using a 75 g OGTT

(Wolever &Mehling, 2002). They were randomly assigned to consume one of

three diets for four months:

1. a diet containing 50–55% carbohydrate using at least one high-GI food at

every meal (high-GI, n¼ 11);

2. a diet containing 50–55% carbohydrate using at least one low-GI food at

every meal (low-GI, n¼ 13); or

3. a diet in which carbohydrate intake was reduced to 40–45% of energy and

monounsaturated fat (MUFA) intake was increased (MUFA, n¼ 11).

Subjects were provided with high- and low-GI foods and food sources of

MUFA to use in their diets. Insulin sensitivity, insulin secretion and disposi-

tion index (the product of insulin sensitivity and insulin secretion) were

measured using a frequently-sampled intravenous glucose tolerance test at

baseline and after four months on the diet. To compare the effects of the three

diets on blood glucose, insulin and free-fatty acid (FFA) responses while

consuming the study diets, subjects also underwent eight-hour metabolic

profiles. At baseline, all subjects consumed breakfast and lunch meals

reflecting the high-GI diet. At four months they consumed breakfast and

lunchmeals reflecting the diet towhich they had been randomized; subjects on

high-GI consumed the same meals as at baseline, subjects on low-GI

consumed meals of the same energy and carbohydrate content but using

low-GI instead of high GI foods, and subjects onMUFA consumedmeals with

the same energy as at baseline but containing less carbohydrate and more

MUFA.

The results showed that disposition index tended to deteriorate on the high-

GI and MUFA diets, but significantly increased on the low-GI diet. The

change in disposition index on the low-GI diet was significantly different from

the changes on both the high-GI and MUFA diets (Figure 10.2).

Similar effects have been seen in other studies. For example, 72 subjects

with metabolic syndrome were randomly assigned to receive a diet containing

oat, wheat and potatoes (foods shown to elicit high glucose and insulin

responses) or a diet containing whole rye breads and pasta (low glucose and


3GCH10 07/20/2013 8:53:9 Page 199

insulin responses). Beta-cell function improved significantly on the rye-pasta

diet compared to the oat-wheat-potato diet (Laaksonen et al., 2005). These

studies suggest that low-GI foods reduce diabetes risk by improving beta-cell

function.

10.6 MECHANISMS BY WHICH LOW-GI FOODSIMPROVE BETA-CELL FUNCTION

Mechanisms by which low-GI foods may increase beta-cell function include

reduced glucose toxicity, reduced serum FFA and increased levels of incretin

hormones such as GLP-1.

10.6.1 Glucose toxicity

Glucose toxicity refers to damaging effects of high blood glucose concentra-

tions per se on body tissues and regulatory processes due to a variety of

mechanisms, including increased flux through the polyol and glucosamine

pathways, increased non-enzymatic glycation products and glycosylation of

proteins, activation of diacylglycerol and protein kinase C and increased

oxidative and carbonyl stress (Brownlee, 2005).

Insulin sensitivity →

Insu

lin s

ecre

tion

→

p = 0.05

p = 0.05

Low GI

High GI

MUFA

Figure 10.2 Changes in insulin sensitivity and insulin secretion in subjects withimpaired glucose tolerance after four months on a high-carbohydrate, high-glycemicindex (GI) diet (high-GI), a high-carbohydrate, low-GI diet (low-GI) and a low-carbohydrate, high-monounsaturated fat (MUFA) diet. Adapted from Wolever andMehling (2002).


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Glucose toxicity has been implicated in the pathogenesis of diabetes by

reducing beta-cell function (Leahy et al., 1988). However, if glucose toxicity

was the only mechanism involved, any method of reducing blood glucose

should have a beneficial effect on beta-cell function. That this is not the case is

shown by the fact that, in subjects with IGT, reducing diet GI improved beta-

cell function, but reducing the amount of carbohydrate consumed did not,

despite the fact that both diets reduced mean eight-hour blood glucose

compared to the high-GI diet and did so to an equivalent extent (Wolever

& Mehling, 2003). Similarly, in subjects with type 2 diabetes a low-GI diet

reduced serum c-reactive protein as a marker of chronic inflammation, while a

lower carbohydrate, high MUFA diet did not (Wolever et al., 2008). Both of

these studies suggest that reducing glucose toxicity is not the only mechanism

by which low-GI foods improve beta-cell function.

10.6.2 Reduced serum free fatty acids (FFA)

The beta-cell secretes insulin in response to a rise in blood glucose, and it has

been proposed that the intracellular mechanism by which glucose stimulates

insulin secretion is via intracellular long-chain fatty acids. A rise in blood

glucose increases glucose entry into the beta-cell and, hence, increases flux

through the glycolytic pathway and glucose oxidation. Increased glucose

oxidation within the beta-cell inhibits the oxidation of fatty acids, causing the

intracellular concentration of fatty acids to rise and this, in turn, stimulates

the release of insulin from the cell (Prentki & Corkey, 1996). This is confirmed

by studies showing that the addition of fatty acids to the medium of cultured

beta-cells increases insulin secretion (Zhou & Grill, 1994) and, in humans

in vivo, acutely increasing the serum FFA concentration increases insulin

secretion (Carpentier et al., 1999).

However, in the presence of chronically high concentrations of intra-

cellular fatty acids and glucose, the glucose signal is lost. Prolonged

exposure of cultured beta cells to high levels of FFA and glucose reduces

insulin secretion (Zhou & Grill, 1994). Similarly, in humans, a prolonged

increase in serum FFA inhibits insulin secretion (Carpentier et al., 1999).

For this reason, we measured serum FFA concentrations after four months

on the low-GI, high-GI and MUFA diets and showed that 0–8 hours mean

serum FFA concentration on the low-GI diet was significantly lower than on

high-GI, whereas the levels of FFA on the MUFA diet were similar to those

on high-GI (Wolever & Mehling, 2003). This suggests that it may be

necessary to reduce both glucose and FFA in order to improve beta-cell

function.


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10.6.3 Increased GLP-1 secretion

GLP-1 (glucagon-like polypeptide-1) is an incretin, secreted from the L-cells

in the lower end of the small intestine, which has multiple biological

functions, including delaying gastric emptying, increasing satiety, increasing

insulin sensitivity and increasing insulin secretion (Drucker, 1998). The latter

is thought to be achieved not only by increasing the amount of insulin secreted

by each beta-cell, but also by increasing the number of beta-cells. GLP-1 is

secreted in response to the presence of nutrients in the distal small intestine

and also via a neuroendocrine loop, whereby GIP secretion in the upper

intestine sends a signal via the vagus nerve and the brain to the L-cells

(Brubaker & Anini, 2003).

Animal studies suggest that GLP-1 secretion is also stimulated by the

short-chain fatty acids (SCFA) produced upon fermentation of dietary fibre

and resistant starch in the large intestine (Reimer & McBurney, 1996).

This might be a mechanism by which diets high in cereal fibre reduce

diabetes risk. The cereal fibre most commonly consumed in North America

is wheat bran; however, studies lasting 4–12 weeks have shown no signifi-

cant effect of wheat fibre on blood glucose control or oral glucose tolerance

(Munoz et al., 1979; Kestin et al., 1990; Jenkins et al., 2002; Costabile

et al., 2008).

One way to reconcile discrepancy between epidemiological studies show-

ing that cereal fibre is a strong predictor of diabetes risk (Table 10.1), and

long-term clinical trials showing that wheat bran has little or no effect on

glucose metabolism, might be that the clinical trials lasted only four weeks to

three months, which is not long enough for the colonic bacteria to adapt to the

change in fibre intake. The colon is a very complex ecosystem, containing

hundreds of species of bacteria. If an input into an established ecosystem (here

the input is the amount of fibre entering the colon) is changed, the output of the

ecosystem becomes unstable and varies with time until a new equilibrium is

reached. The more complex the ecosystem, the longer it takes for a new

equilibrium to be established (Feng & Chai, 2008).

Studies in animals demonstrate this concept. For example, in a four-month

study, when the resistant starch content of the diet of rats was increased, the

SCFA content of the caecum began to increase and continued to increase over

entire period of the study (Le Blay et al., 1999). This suggests that it takes the

colonic ecosystem more than four months to equilibrate after a change in fibre

intake. Thus, we hypothesized that cereal fibre, in the form of All-Bran1

cereal would significantly increase serum SCFA concentrations and GLP-1

secretion, but that this would only be apparent after a long adaptation period of

at least six months.


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To test this, we randomly assigned 28 hyperinsulinemic subjects to

consuming two cups of a low fibre cereal per day, or one cup of All-

Bran1 daily for one year (Freeland et al., 2010). At baseline, and after every

three months, subjects underwent a metabolic profile in which the glucose,

insulin, FFA, SCFA and GLP-1 responses elicited by the diet were measured.

The results showed significant time� treatment interactions for serum ace-

tate, butyrate and GLP-1; that is, the difference between the diets depended

upon the time. Acetate increased temporarily after nine months on the diet,

butyrate began to increase at three months and reached a plateau after nine

months, and GLP-1 tended to gradually increase with time to reach a level

significantly higher than the control after 12 months. These results supported

the hypothesis that cereal fibre may reduce diabetes risk by increasing GLP-1

secretion mediated by colonic short chain fatty acids, but that these events take

many months to occur, as the colonic ecosystem gradually adapts to the

increased fibre intake.

10.7 CONCLUSIONS

The quality of dietary carbohydrates may be an important factor in determin-

ing risk for type 2 diabetes. The results of recent studies suggest that a

deterioration in beta-cell function may be the critical pathophysiological event

which causes type 2 diabetes. Evidence has been presented here that low-GI

carbohydrates may preserve beta-cell function by reducing postprandial

glucose fluctuations and, hence, glucose toxicity, while at the same time

reducing serum FFA concentrations. Cereal fibre may preserve beta-cell

function, at least in part, by increasing colonic fermentation and up-regulating

GLP-1 secretion, but these effects may not occur until the colon has adapted to

the increased intake of fibre – a process which may take 6–9 months or more.

However, whether low-GI foods or cereal fibre will actually prevent type 2

diabetes in humans remains to be shown.

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11 Resistant Starch on Glycemiaand Satiety in Humans

Mark D. HaubDepartment of Human Nutrition, Kansas State University, USA

11.1 INTRODUCTION

There is a major public health effort to decrease the prevalence of obesity-

related health outcomes. Given the role of diet and nutrition on the aetiology

of obesity, it is imperative that dietary options should be targeted to assist with

preventing these comorbid conditions such as type 2 diabetes, dyslipidaemia

and other cardiometabolic complications.While resistant starch (RS) has been

studied by cereal chemists and food companies for several years, studies

conducted by clinical scientists have been limited, but these are increasing and

providing interesting results indicating a strong evidence that RS may provide

significant metabolic health benefits. The majority of studies indicates a

capacity to lower glycemia and improve insulin sensitivity. Other data, while

less conclusive, indicate the potential to attenuate satiety and decrease

subsequent food intake. Thus, there seems to be an inverse relationship

between glycemic response and food intake following the consumption of

RS or foods containing adequate amounts of RS. Moreover, while the data are

limited, evidence seems to indicate that different types of RS can elicit

different metabolic responses.

This review will focus on the glucoregulatory aspects of RS, with discus-

sions on outcomes from primarily human clinical trials relating to satiety, food

intake and potential to address the obesity issue present in most industrialized

societies.

207




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11.2 DIET AND RESISTANT STARCH

Diabetes, obesity and cardiovascular disease are interrelated. Diet is one

factor that links them all together. Specifically, the current obesity epidemic

has been suggested to be a result of increased carbohydrate intake (Gaesser,

2007), even though dietary fat has frequently been touted as the primary

dietary culprit of many deleterious metabolic conditions. The fact is that we,

as a nation, actually increased carbohydrate consumption – perhaps to

address the public health message to avoid dietary fat – during the latter

years of the past century, just as the obesity epidemic began to surge

(Gaesser, 2007). Thus, the national response to decrease fat may have

inadvertently led to overconsumption of carbohydrates. From one perspec-

tive, the public health effort to decrease the percent of energy derived from

fat in our diets was moderately successful; however, total energy intake and

attaining a weight-maintaining energy balance was not affected by those

behaviours.

So, while it is laudable to try to change the behaviour of individuals (to

choose other foods), it might be more effective to address this nutritional issue

by providing bioactive compounds that ‘behave’ like traditional starch, yet

elicit a more favourable metabolic profile. In other words, let people continue

to choose the foods they prefer, but simply make those foods healthier by

incorporating bioactive ingredients and/or ingredients that affect energy

metabolism. This might seem to be an achievable goal, but some past efforts

have not proven to be acceptable to consumers.

To address this issue, RS shows promise at successfully improving

glucoregulation in a consumer-acceptable fashion, while eliciting side effects

that are similar to typical dietary fibres (e.g. bran) that have had difficulty

being accepted by a most consumers. Compared with typical monosacchar-

ides and disaccharides, RS elicits minimal glucose and insulin excursions and

can be incorporated easily into regular food items with general acceptance by

consumers, which is a barrier to many dietary fibres. That said, there is a

paucity of evidence, especially from humans, to illustrate the glucose-low-

ering mechanisms. The common theory is based on the glucose-fatty acid

cycle, whereby increasing fat oxidation allows for increased insulin sensitivity

and glucose disposal (Randle et al., 1963).

Testing this theory in human clinical trials will increase our understanding

of the mechanisms through which RS, and other modified starches with

similar properties, elicit their benefits. This will help food and pharmaceutical

scientists to develop and/or enhance products to better prevent, manage and

treat metabolic conditions. Currently, evidence indicates a glucoregulatory

benefit, but how this occurs is not well established.


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11.3 RESISTANT STARCH AND INSULIN SENSITIVITY

RS is a dietary carbohydrate that has been frequently studied. Most RS

research, however, has focused on fermentation characteristics, while few

studies have been designed to investigate mechanisms involved with increas-

ing insulin sensitivity. Most of the mechanistic studies have used rodents and

other small animals rather than using a human clinical trial.

Basing RSmechanisms on animal study outcomes is problematic, given the

coprophagic tendency of some species, whichmakes it difficult to interpret the

results and apply those potentially confounded results to humans. Moreover,

humans prefer eating a varied diet throughout the day and over time, while

animal studies test ingredients with the animals eating the same food at every

meal, every day. An additional research design and translational science issue

is that we do not fully understand how to relate animal outcomes to humans,

given that the dose and duration of treatment given to animals may not apply

to typical human scenarios.

There has been one human clinical trial (Robertson et al., 2005) that

specifically investigated the effects of resistant starch on insulin sensitivity

using the standard euglycaemic-hyperglycaemic clamp. They observed that

individuals consuming 30 g/day of RS over four weeks elicited significant

increase in glucose disposal and decreased insulin area under the curve during

a meal tolerance test. However, while they observed increased insulin

sensitivity, they were unable to discern clearly how RS elicited this response.

Results from other human clinical trials (Yamada et al., 2005; Robertson

et al., 2003; Haub et al., 2010; Behall et al., 2006; Al-Tamimi et al., 2010),

support this notion that RS decreases postprandial glucose and insulin

responses. While that evidence is fairly strong and consistent, the long-

term benefits of RS are still not well established. What is less clear is the

mechanism through which long-term RS ingestion exerts its benefits, and

whether RS consistently affects health and lifestyle outcomes (e.g. obesity,

satiety, and food intake) other than glucose metabolism in humans.

11.4 CURRENT THEORETICAL MECHANISM

A testable theory to explain the metabolic regulation by RS pertains to the

formation of short chain fatty acids via fermentation, which subsequently

increases fat oxidation (centrally and peripherally). While this theory has not

formally been tested relative to RS, it is supported by the observation that

increasing fat oxidation is a key factor to increase insulin sensitivity in those

with insulin resistance (Figure 11.1).

Resistant Starch on Glycemia and Satiety in Humans 209

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Moreover, it was reported by Higgins et al. (2004) that the ingestion of RS

leads to acute increases in lipid oxidation, which may lead to decreased fat

accumulation and/or accretion over time. While the role of short chain fatty

acids in this process is plausible, inducing an energy deficit also increases fat

oxidation and insulin sensitivity by decreasing the inhibition on fat oxidation

elicited by insulin and other glucoregulatory hormones. Thus, since RS only

yields half the energy of regular starch (�2 kcal/g), it is plausible that a lower

energy state of the cell or tissue may increase fat oxidation.

Specifically, based on the outcomes of Robertson et al. (2003), it was

suggested that the increased insulin sensitivity arises from alterations in

substrate oxidation due to the increased availability of short chain fatty acids

derived from fermentation of RS (Figure 11.1). However, these researchers

failed to include a treatment that only increased short chain fatty acids,

thereby limiting their capacity to better understand how RS elicits the ‘second

meal effect’ or whether other factors, such as an energy deficit, were

contributing factors.

However, in a study by Johnston et al. (2010), the RS treatment (40 g/d)

over 12 weeks elicited increased insulin sensitivity without a concomitant

change in body weight or intracellular lipid content. These data indicate that

Resistant starch

Fermentation

Improved metabolic health

Energy deficit‘Weight loss’

Short chain fatty acids

blood glucoseand insulin

?

?

Figure 11.1 This figure depicts means through which RS is suggested to elicitmetabolic benefits. It has been established that RS decreases blood glucose andinsulin, which decreases risk for type 2 diabetes over time. It has, also, been shown tobe fermented by gut microbiota, thereby changing the gut ecology and producingproducts (short chain fatty acids) that have been suggested to improve metabolichealth. To date, however, it is not knownwhether some of the changes attributed to RSare due to changes in energy balance as RS may decrease energy intake acutely and/or chronically. Thus, some benefits attributed to RS may be indirect due to decreasedenergy being absorbed, and/or different ratios of macronutrients due to conversionof some RS into fatty acids by the gut microbiota.


CH11GR 07/29/2013 16:40:56 Page 211

RS may even improve glycemia and insulinaemia, regardless of energy intake

or lipid metabolism.

11.5 SATIETY

OneuniqueeffectofRSthatseemstobedevelopingistheimpactthatthesestarches

seemtohaveonsatiety.With lifestylemodification–specifically reducingenergy

intake – being a regular recommendation to prevent and treat various metabolic

diseases and conditions (Poirier et al., 2006; Jakicic et al., 2001), this bioactive

effect could be extremely beneficial from a public health perspective.

The means through which RS has been shown to induce satiety and lead to

decrease energy intake (may not decrease volume or weight of food con-

sumed) seems likely to involve alterations in PYY and other neuroendocrine

components (Zhou et al., 2008). Zhou et al. (2008) administered RS to mice

over 32 days and observed that active forms of PYYand GLP-1 increased. The

mice eating RS also gained less fat mass and had a lower ratio of body fat to

body weight, while eating the same weight of food.

Similarly, So et al. (2007) fed mice high and low RS diets and measured

numerous body composition and metabolic parameters after the eight-week

intervention. They observed several favourable outcomes following the high

RS treatment, compared with the low RS:

1. decreased intracellular fat deposition;

2. decreased body weight and adiposity;

3. smaller adipocytes;

4. their brain imaging results indicated greater satiety despite decreased

energy intake.

While those studies were completed in mice, the satiating effect of RS has

been reported in humans. Although hormone levels were not reported, Willis

et al. (2009) observed satiety and feelings of fullness were evident longer

following trials with RS and corn bran, compared with lower fibre bleached

oat bran, b-glucan, and polydextrose. In support with those satiety results,

outcomes from Anderson et al. (2010) indicate that foods containing 40–70%

RS seem to reduce food intake during meals later in the day. In a study out of

our lab (Haub et al., 2012), RS4 from potato starches elicited similar ratings of

satiety when ingested with an energy dense beverage (dextrose solution) or

ingested with water. Taken together, the mechanisms for the reduced intake

and ratings of satiety are not well understood, as gut hormone responses have

either not been studied or did not elicit consistent results.


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11.6 FERMENTATION ANDGUT MICROBIOTA

A new area of research has been better in understanding how gut microbiota

(especially lactobacilli and bifidobacteria), fermentation, and short chain

fatty acids impact human health (Roberfroid et al., 2010), with a few of these

aspects depicted in Figure 11.1. The food industry has been actively placing

prebiotic and probiotic foods on the market to provide consumers with foods

to try to meet this health outcome. Specific to RS, Martinez et al. (2010)

reported changes to gut microbiota following the ingestions of RS2 and RS4

by human volunteers. They observed that RS intake significantly altered gut

microbiota in as little as one to three weeks, and that RS2 and RS4 elicited

different changes in microbiota.

Moreover, short chain fatty acids (SCFA) formed from the fermentation of

dietary fibre and RS sources have been selected as potential factors for

increased satiety. However, a study using oligosaccharides (Hess et al.,

2010), reported that increased fermentation does not necessarily alter satiety

in men or women. Collectively, RS and the by-products of fermentation may

interact synergistically to elicit feelings of fullness. This is an area requiring

more research to understand better how the effects of gut changes in bacteria

and alterations in the fermentation process affect human health acutely and

over time.

11.7 EFFECT OF RS TYPE

An area of RS research that needs more investigation is comparing the forms

and types of RS currently available. Specifically, there is a paucity of data

comparing RS compounds of different types (e.g. Type 2 vs. Type 3) within

the same trial to determine similarities and differences on in vivo outcomes.

This information is imperative for consumers and others interested in select-

ing RS to incorporate into foods and diets.

To our knowledge, there is only one peer-reviewed paper to have compared

commercially available RS types from different companies on glycemia.

Haub et al. (2010) compared the glucose lowering effects of a commonly

tested type of RS (RS2) with a less studied cross-linked type (RS4). It was

observed that the RS4 elicited a greater reduction in capillary blood glucose

than RS2. This study clearly indicated that the type of RS used in clinical

studies needs to be accounted for, since different types can elicit significantly

different responses.

Together with the results fromMartinez et al. (2010), it can be erroneous to

conclude that all RS types will elicit the same physiological or psychological


CH11GR 07/29/2013 16:40:56 Page 213

responses. Further research investigating the differences in RS types will help

illustrate how much of each RS is necessary to elicit a desired response. This

outcome is critical when developing recipes and/or budgets for individual

consumers or companies.

11.8 SUMMARY

Over the past two decades, data from animal and human intervention studies

demonstrate that RS seems to elicit favourable changes in glucoregulatory

outcomes and factors relating to gut health. The results from animal studies

indicate a clearer effect on body composition and satiety, while the human

data are not as conclusive in that regard. More studies designed to test body

composition effects are needed to enhance our understanding of RS as a

potential ingredient to significantly affect obesity-related outcomes. Likewise,

while it is apparent that RS, as with other dietary fibres, does elicit changes in

metabolic and gut health outcomes, the appropriate dose and/or RS-contain-

ing foods that elicit desired outcomes and significant health effects are still not

well known.

REFERENCES

Al-Tamimi, E.K., Seib, P.A., Snyder, B.S., Haub, M.D. (2010). Consumption of Cross-Linked Resistant Starch (RS4(XL)) on Glucose and Insulin Responses in Humans.Journal of Nutrition and Metabolism Article ID 651063, 6p.

Anderson, G.H., Cho, C.E., Akhavan, T., Mollard, R.C., Luhovyy, B.L., Finocchiaro, E.T.(2010). Relation between estimates of cornstarch digestibility by the Englyst in vitromethod and glycemic response, subjective appetite, and short-term food intake in youngmen. American Journal of Clinical Nutrition 91, 932–939.

Behall, K.M., Scholfield, D.J., Hallfrisch, J.G., Liljeberg-Elmstahl, H.G. (2006). Con-sumption of both resistant starch and beta-glucan improves postprandial plasma glucoseand insulin in women. Diabetes Care 29, 976–981.

Gaesser, G.A. (2007). Carbohydrate quantity and quality in relation to body mass index.Journal of the American Dietetic Association 107, 1768–1780.

Haub,M.D., Hubach, K.L., Al-Tamimi, E.K., Ornelas, S., Seib, P.A. (2010). Different typesof resistant starch elicit different glucose responses in humans. Journal of Nutrition andMetabolism Article ID 230501.

Haub, M.D., Louk, J.A., Lopez, T.C. (2012). Novel resistant potato starches on glycemiaand satiety in humans. Journal of Nutrition and Metabolism Article ID 478043, 4p.

Hess, J.R., Birkett, A.M., Thomas, W., Slavin, J.L. (2010). Effects of short-chainfructooligosaccharides on satiety responses in healthy men and women. Appetite56(1), 128–34.


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Higgins, J.A., Higbee, D.R., Donahoo W.T., Brown, I.L., Bell, M.L., Bessesen, D.H.(2004). Resistant starch consumption promotes lipid oxidation. Nutrition &Metabolism1, 8.

Jakicic, J.M., Clark, K., Coleman, E., Donnelly, J.E., Foreyt, J., Melanson, E., Volek, J.,Volpe, S.L. (2001). American College of Sports Medicine position stand. Appropriateintervention strategies for weight loss and prevention of weight regain for adults.Medicine and Science in Sports and Exercise 33, 2145–2156.

Johnston, K.L., Thomas, E.L., Bell, J.D., Frost, G.S., Robertson, M.D. (2010). Resistantstarch improves insulin sensitivity in metabolic syndrome. Diabetic Medicine 27,391–397.

Martinez, I., Kim, J., Duffy, P.R., Schlegel, V.L., Walter, J. (2010). Resistant starches types2 and 4 have differential effects on the composition of the fecal microbiota in humansubjects. PLoS ONE 5(11): e15046.

Poirier, P., Giles, T.D., Bray, G.A., Hong, Y., Stern, J.S., Pi-Sunyer, F.X., Eckel, R.H.(2006). Obesity and cardiovascular disease: pathophysiology, evaluation, and effect ofweight loss: an update of the 1997 American Heart Association Scientific Statement onObesity and Heart Disease from the Obesity Committee of the Council on Nutrition,Physical Activity, and Metabolism. Circulation 113, 898–918.

Randle, P.J., Garland, P.B., Hales, C.N., Newsholme, E.A. (1963). The glucose fatty-acidcycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.Lancet 1, 785–789.

Roberfroid, M., Gibson, G.R., Hoyles, L., McCartney, A.L., Rastall, R., Rowland, I.,Wolvers, D., Watzl, B., Szajewska, H., Stahl, B., Guarner, F., Respondek, F., Whelan, K.,Coxam, V., Davicco, M.J., L�eotoing, L., Wittrant, Y., Delzenne, N.M., Cani, P.D.,Neyrinck, A.M., Meheust, A. (2010). Prebiotic effects: metabolic and health benefits.British Journal of Nutrition 104(Suppl 2), S1–63.

Robertson, M.D., Currie, J.M., Morgan, L.M., Jewell, D.P., Frayn, K.N. (2003). Prior short-term consumption of resistant starch enhances postprandial insulin sensitivity in healthysubjects. Diabetologia 46, 659–665.

Robertson, M.D., Bickerton, A.S., Dennis, A.L., Vidal, H., Frayn, K.N. (2005). Insulin-sensitizing effects of dietary resistant starch and effects on skeletal muscle and adiposetissue metabolism. American Journal of Clinical Nutrition 82, 559–567.

So, P.W., Yu, W.S., Kuo, Y.T., Wasserfall, C., Goldstone, A.P., Bell, J.D., Frost, G. (2007).Impact of resistant starch on body fat patterning and central appetite regulation. PLoSONE 2, e1309.

Willis, H.J., Eldridge, A.L., Beiseigel, J., Thomas, W., Slavin, J.L. (2009). Greater satietyresponse with resistant starch and corn bran in human subjects. Nutrition Research 29,100–105.

Yamada, Y., Hosoya, S., Nishimura, S., Tanaka, T., Kajimoto, Y., Nishimura, A., Kajimoto,O. (2005). Effect of bread containing resistant starch on postprandial blood glucoselevels in humans. Bioscience, Biotechnology and Biochemistry 69, 559–566.

Zhou, J., Martin, R.J., Tulley, R.T., Raggio, A.M., McCutcheon, K.L., Shen, L., Danna, S.C., Tripathy, S., Hegsted, M., Keenan, M.J. (2008). Dietary resistant starch upregulatestotal GLP-1 and PYY in a sustained day-long manner through fermentation in rodents.American Journal of Physiology – Endocrinology and Metabolism 295, E1160–1166.


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12 The Acute Effects of Resistant Starchon Appetite and Satiety

Caroline L. Bodinham and M. Denise RobertsonDepartment of Nutritional Sciences, Faculty of Health andMedical Sciences, University of Surrey, UK

12.1 APPETITE REGULATION

Appetite is defined as the desire or physical craving to eat and is typically

separated into hunger, satiation and satiety. In a review byMattes et al. (2005),

hunger is defined as ‘sensations that promote food consumption’, satiation as

‘sensations that govern meal size and duration’ and satiety as ‘sensations

that determine the inter-meal period of fasting’. Therefore, all are important

for determining energy intake. Appetite is difficult to quantify, as it varies

greatly between individuals. It is closely regulated by physiological processes,

but it can also be influenced by many other aspects, including psychological

factors (e.g. learned habits) and emotional factors (e.g. as a response to stress),

as well as by external cues (e.g. accessibility of food, the presence or absence

of other individuals or the hedonic properties of the food itself).

The hypothalamus plays a vital role in appetite regulation and the subsequent

control of food intake; it receives peripheral signals from the digestive tract

(gut peptides) and adipose tissue (through signalling molecules, including

leptin and insulin) (Murphy & Bloom, 2004; Wynne et al., 2005). While the

hypothalamus is composed of many areas (nuclei), the arcuate nucleus (ARC)

is thought to be the most important in appetite regulation and is essential for

interpreting these peripheral signals (Wynne et al., 2005; Dhillo, 2007). The

ARC has an incomplete blood-brain barrier, so peripheral signals are able to

cross the barrier, interact with receptors and consequently cause the release of

neuropeptides that subsequently regulate ingestion (Neary et al., 2004).

There are two classes of neurons in the ARC: those that stimulate

and those that inhibit food intake. The neurons that stimulate appetite

(and, therefore, promote food intake) are neuropeptide Y (NPY) and

215




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agouti-related peptide (AgRP) (Wynne et al., 2005). NPY causes an increase

in food intake through activation of the G-protein-coupled receptors Y1 and

Y5, while AgRP causes an increase in food intake through antagonism of

the melanocortin-4 (MC4) receptor and, therefore, the inhibition of

a-melanocyte-stimulating hormone (a-MSH) (Neary et al., 2004; Wynne

et al., 2005). The neurons that inhibit food intake are pro-opiomelanocortin

(POMC) and cocaine- and amphetamine-regulated transcript (CART). These

neurons stimulatea-MSH,which interacts with theMC4 receptor (Murphy&

Bloom, 2004; Neary et al., 2004; Wynne et al., 2005) that promotes sub-

sequent anorexigenic activity. Once the peripheral signals are interpreted in

the ARC, this can both inhibit and stimulate the different classes of neurons

whether to promote food intake or to prevent further intake.

The peripheral signals from the digestive tract are hormones that are both

anorexigenic (increase satiety) and orexigenic (promote hunger). The hor-

mone ghrelin is unique in being orexigenic, and it is often termed the

‘hormone of hunger’. Anorexigenic hormones are released postprandially

in response to ingested nutrients; they act by both direct and indirect (via the

vagus) mechanisms and promote feelings of satiety. These hormones include

glucagon-like peptide-1 (GLP-1) and peptide YY (PYY).

12.2 MEASUREMENT OF APPETITE IN HUMANS

Appetite is particularly difficult to directly measure in humans, as it is a

subjective sensation that can be influenced by many factors. Human studies,

therefore, have used different indirect measures in order to assess appetite

sensations (Mattes et al., 2005). The most frequently used indirect methods

are recording food intake, using questionnaires regarding appetite feelings and

measuring biomarkers (Mattes et al., 2005).

Records of food intake rely on participants accurately recording all they

have to eat and drink over a certain time period, as well as on accurate

interpretation by the investigators. As such, food records may not always

provide precise results. Food intake can also be assessed at weighed and

controlled ad libitum meals. While these do provide accurate measures of

food intake, they might not show habitual intakes, as they are often completed

in artificial environments.

Questionnaires relating to subjective appetite feelings are often used in

human studies. These questionnaires rely on those filling out the questions

reporting on their thoughts and feelings relating to hunger, satiation and

satiety. Typically, there are two main types of questionnaire that are used –

category scales and visual analogue scales (VAS). These are quick and easy to


3GCH12 07/20/2013 9:50:29 Page 217

complete and have been proven to be reproducible and valid in appetite

research.

Biomarkers of appetite are also often measured in human studies, and

include changes to gut hormone concentrations. These are often a preferred

measurement of changes to appetite, as they are less likely to be influenced by

other factors.

12.3 PROPOSED MECHANISMS FOR AN EFFECT OFRESISTANT STARCH ON APPETITE

Although the exact mechanism as to how resistant starch (RS) may affect

appetite is not known, various mechanisms have been proposed. These

mechanisms may be specific to RS or may be ubiquitous for all dietary fibres.

As RS is not digested in the small intestine but is fermented in the colon, the

most likely mechanism behind an effect on appetite is due to fermentation and

the subsequent increase in production of short-chain fatty acids (SCFA). The

SCFA may increase production of GLP-1 and PYY which, in turn, may

activate the neurons in the hypothalamus, therefore affecting behaviour and

causing changes to food intake (Figure 12.1).

The cells that secrete GLP-1 and PYY are primarily located in the ileum

and colon. GLP-1 receptors have been located in the hypothalamus (Murphy

& Bloom, 2004; Holst, 2007), therefore suggesting a role for GLP-1 in

directly affecting food intake. This has been shown in rodent models, where

central infusion of GLP-1 reduced food intake (Turton et al., 1996). GLP-1

may also indirectly affect food intake via the vagus nerve, as studies in rodents

Resistant starch

consumption

Increased SCFA

Increased GLP-1 and

PYY

Activation ofneurons in

ARC

Modifiedbehaviour/appetite

Decreasedfood intake

Figure 12.1 Possible pathway by which resistant starch consumption affects appetiteand food intake.

The Acute Effects of Resistant Starch on Appetite and Satiety 217

3GCH12 07/20/2013 9:50:29 Page 218

have shown that, following a vagotomy, the anorexic effects of GLP-1 are lost

(Dhillo, 2007). Studies where GLP-1 has been infused in humans provide

mixed results, with some studies showing an effect (Flint et al., 1998; Naslund

et al., 1999) and others none (Long et al., 1999; Naslund et al., 1998).

However, a meta-analysis (which included the above studies in addition to

others) showed a dose-dependent reduction in food intake following

GLP-17–36 (biologically active form) peripheral administration in humans

(Verdich et al., 2001).

Similarly, studies that have infused PYY in rodents and humans have found

a reduction in food intake (Batterham et al., 2002; Challis et al., 2003). In one

instance, the decrease in energy intake was 30% following peripheral infusion

of the active form (PYY3–36) in humans (Batterham et al., 2003). PYY may

also be released due to neural reflex, potentially mediated by the vagus nerve

(Fu-Cheng et al., 1997).

It has also been shown that while these hormones can reduce food intake

independently, when they are administered together, the effect on reducing

food intake is additive (Neary et al., 2005; Steinert et al., 2010).

Therefore, as these two hormones have been shown to reduce food intake,

they are likely to be the most affected by RS consumption due to fermentation

in the colon.

12.4 RODENT DATA

Results from rodent studies have shown promising effects of RS intake on

appetite and food intake, as well as on gut hormone regulation. However, these

rodent studies have not been acute (10–65 days), so therefore the translation of

the data from these experiments to acute affects in humans is not possible. As

these studies do provide some evidence for potential mechanisms, they will be

discussed briefly below and are summarised in Table 12.1.

Based on the potential pathway by which RS may affect appetite and food

intake described above (Figure 12.1), there is evidence in rodent models for

each of the steps as shown in Figure 12.2 and discussed in more detail below.

A few experiments in rodents have shown increased concentrations of

SCFA following RS consumption in studies relating to appetite work, as well

as effects of the RS consumption on changes to gene expression. Keenan et al.

(2006) proved significantly higher caecal SCFA concentrations following a

RS diet compared to a non-fermentable cellulose energy controlled diet; they

also observed increased PYY and proglucagon (the precursor molecule for

GLP-1) gene transcription in the caecum and large intestine following RS

compared to the control. Zhou et al. (2006) showed that, after a RS diet,


3GCH12 07/20/2013 9:50:29 Page 220

compared to control, PYY and proglucagon mRNA expression were signifi-

cantly unregulated in the caecum and colon. Further work by Zhou et al.

(2008) also showed in vitro that the fermentation of RS increased the

concentrations of SCFA, and that the SCFA (when at concentrations similar

to those observed in the caecal content in the study by Keenan et al. (2006))

directly stimulated proglucagon gene expression.

Several studies in animals have shown increased GLP-1 and PYY concen-

trations following RS consumption. The study by Keenan et al. (2006) found

greater serum PYY and GLP-1 concentrations following the RS diet, but no

effect on serum ghrelin or cholecystokinin (CCK) concentrations. Plasma

PYYand GLP-1 concentrations were also increased in the study by Shen et al.

(2009). Zhou et al. (2008) found RS resulted in significantly higher plasma

GLP-1 and PYY concentrations throughout the day. Aziz et al. (2009)

compared high-amylose with high amylopectin diets and found that the

high-amylose diets resulted in higher plasma GLP-1 and PYY concentrations.

There is also evidence from rodent studies for an effect of RS consumption

on activation of neurons in the hypothalamus. Shen et al. (2009) found that,

following a high RS diet, POMC expression in the ARC was significantly

upregulated, but there was no effect on NPY or AgRP. So et al. (2007)

conducted manganese-enhanced magnetic resonance imaging during ad

libitum food intake to monitor neuronal activity in the areas of the hypo-

thalamus involved in appetite regulation and found that there appeared to be a

satiating effect of the RS diet compared to the rapidly digestible starch.

Most of these studies, in the course of looking at possible mechanisms

behind an effect of RS on appetite, have also reported changes to food intake

and weight, with mixed findings. Aziz et al. (2009) reported that the high-

amylose diets, when consumed ad libitum, resulted in lower energy intakes,

weight gain and fat pad mass. Similarly, So et al. (2007) also reported lower

Resistant starch

consumption

Increased SCFA

Increased GLP-1 and

PYY


ARC



Increased gene

expression in caecum and

colon

Lower body weight and

fat mass

Figure 12.2 Possible pathway by which resistant starch consumption affects foodintake and behaviour in rodents.


3GCH12 07/20/2013 9:50:29 Page 221

energy intakes in the RS group and found that, although body weight was the

same in both groups, the RS group had lower total, subcutaneous, visceral and

liver fat, and also smaller adipocytes. However, the studies by Keenan et al.

(2006), Zhou et al. (2008) and Shen et al. (2009) found similar food intakes

between groups, but lower body weights and fat mass in the RS group.

From these studies in rodents, it is clear that high RS intakes result in

increases in both GLP-1 and PYY (satiety hormones), most likely due to

increased production of SCFA from fermentation. Although, in some of these

studies, RS appears to decrease both body weight and fat mass, the effects on

actual energy and food intake are less clear.

In many of these animal experiments, the amount of RS given would be

much greater than would normally be consumed by a human. Similarly, the

length of time (normally days to weeks) that the rodents were fed the starch is

difficult to compare to the same time span in humans. The gut physiology of

rodents is also very different (greater large bowel length) from that of humans

and, therefore, fermentation effects in a rodent can not be directly translated

into similar effects to be observed in humans.

While the evidence for an effect of RS on appetite in rodent studies is

positive, these results have yet to be demonstrated fully in humans.

12.5 HUMAN DATA

Compared to the data from rodent studies, the acute effects (up to 48 hours) of

RS on appetite are less clear in humans.

There are few human studies that have directly investigated the effects of

RS on appetite and food intake, with these studies providing mixed findings

(summarised in Table 12.2). Direct comparisons are hampered by the use of

different protocols and different types of RS (the majority of these studies

used type 2 or 3). Some early studies have also compared the different effects

of amylose and amylopectin on appetite and food intake. As RS is high in

amylose, the results from these studies are included.

Based on the potential pathway by which RS may affect appetite and food

intake described above (Figure 12.1), there is only some evidence in humans for

some of the steps, as shown in Figure 12.3 and discussed in more detail below.

Very few human studies have reported the effects of RS on GLP-1 and

PYY. In the study by Raben et al. (1994) it was found that there were lower

plasma GLP-1 concentrations with RS, compared with their placebo.

Bodinham et al. (2013) also found lower GLP-1 concentrations following

a breakfast containing RS compared to a placebo. A study by Klosterbuer et al

(2012) found no effect of RS on GLP-1 concentrations. Although a study by


3GCH12 07/20/2013 9:50:30 Page 224

Robertson et al. (2003) was not investigating the effects of RS on appetite, one

of their outcomes was the effects of RS consumption on GLP-1 concentra-

tions, and this also showed no effect. The lack of evidence for an effect on gut

hormones in humans could be due to the neuronal effects of GLP-1 and PYY

which would not be measured in the general circulation. Alternatively, as

GLP-1 is rapidly broken down in the circulation by dipeptidyl peptidase IV

(DPP-IV) and both hormones are quickly cleared from the blood via the liver

(Holst, 2007), the hormones may not appear in peripheral blood at raised

concentrations, but there may be raised concentrations in splanchnic blood.

Several studies have measured the effects of RS on modifying appetite

using changes to subjective questionnaires. These studies have also reported

mixed findings:

� three studies have reported no differences between RS/high-amylose and a

control (Weststrate & van Amelsvoort, 1993; Bodinham, et al., 2010;

Klosterbuer et al. 2012);� one study reported lower satiety with RS compared to a control (Raben

et al., 1994);� several studies have reported greater satiety with RS (Kendall et al., 2010;

Willis et al., 2009; Nilsson et al., 2008; Liljeberg et al., 1999; Holt & Brand

Miller, 1995; Granfeldt et al., 1994; van Amelsvoort & Weststrate, 1992).

However, some of these studies also found that the RS supplement was less

palatable than the control used (Kendall et al., 2010; Willis et al., 2009; van

Amelsvoort &Weststrate, 1992), which could account for the effects observed.

Only a few studies have investigated the effects of RS on actual food intake.

Two of these studies (Kendall et al., 2010; Klosterbuer et al., 2012) found no

Resistant starch

consumption

Increased SCFA

Increased GLP-1 and

PYY


ARC



Increased gene

expression

Lower body weight and

fat mass

Figure 12.3 Possible pathway by which resistant starch consumption affects foodintake and behaviour in humans. Circles show areas where there is no acute evidencein human work.


3GCH12 07/20/2013 9:50:30 Page 225

significant differences between treatments for energy intake at the ad libitum

meal. However, two other studies (Bodinham et al., 2010; Holt & BrandMiller,

1995) found a reduced intake with RS. The study by Bodinham et al. (2010)

found the reduced energy intake at both an ad libitum test meal (seven hours

postprandially) and over a 24-hour period. Holt & Brand Miller (1995) found

that intake (in grams) following the high-amylose rice cake was significantly

lower after two hours and over the rest of the day, compared to the low-amylose

rice cake. However, the differences in energy were not significant.

Based on the evidence from the studies using RS and differences in

amylose/amylopectin ratios, there is an acute effect of RS on appetite. In the

few studies where GLP-1 concentrations have been measured (Raben et al.,

1994, Robertson et al., 2003) in human studies, RS does not appear to have

an effect. As the data in rodents suggest that effects on satiety hormones may

be a mechanism by which RS affects appetite, satiety and food intake,

further investigations in humans would be required. Further studies with

similar study designs are also required to determine the dose that is needed

for an effect to occur, as well as other possible mechanisms behind

the effects.

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Batterham, R.L., Cowley, M.A., Small, C.J., Herzog, H., Cohen, M.A., Dakin, C.L., Wren,A.M., Brynes, A.E., Low, M.J., Ghatei, M.A., Cone, R.D., Bloom, S.R. (2002). Guthormone PYY3–36 physiologically inhibits food intake. Nature 418, 650–4.

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Bodinham, C.L., Frost, G.S., Robertson, M.D. (2010). Acute ingestion of resistantstarch reduces food intake in healthy adults. British Journal of Nutrition 103,917–22.

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Challis, B.G., Pinnock, S.B., Coll, A.P., Carter, R.N., Dickson SL., O’Rahilly, S. (2003).Acute effects of PYY3–36 on food intake and hypothalamic neuropeptides expression inthe mouse. Biochemical and Biophysical Research Communications 311, 915–9.

Dhillo, W.S. (2007). Appetite regulation: an overview. Thyroid 17(5), 433–45.Flint, A., Raben, A., Astrup, A., Holst. J.J. (1998). Glucagon-like peptide 1 promotessatiety and suppresses energy intake in humans. Journal of Clinical Investigation 101,515–20.


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Granfeldt, Y., Liljeberg, H., Drews, A., Newman, R., Bjorck, I.M. (1994). Glucose andinsulin responses to barley products: influence of food structure and amylose-amylo-pectin ratio. American Journal of Clinical Nutrition 59, 1075–82.

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Holt, S.H.A., Brand Miller, J. (1995). Increased insulin responses to ingested foods areassociated with lessened satiety. Appetite 24, 43–54.

Keenan, M.J., Zhou, J., McCutcheon, K.L., Raggio, A.M., Bateman, H.G., Todd, E., Jones,C.K., Tullet, R.T., Melton, S., Martin, R.J., Hegsted, M. (2006). Effects of resistantstarch, a non-digestible fermentable fibre, on reducing body fat. Obesity 14, 1523–34.

Kendall, C.W.C., Esfahani, A., Sanders, L.M., Potter, S.M., Vidgen, E. (2010). The effectof a pre-load meal containing resistant starch on spontaneous food intake and glucoseand insulin responses. Journal of Food Technology 8, 67–73.

Klosterbuer, S.S., Thomas , W., Slavin, J.L. (2012). Resistant starch and pullulan reducepostprandial glucose, insulin and GLP-1, but have no effect on satiety in healthy humans.Journal of Agricultural and Food Chemistry 60(48), 11928–11934.

Liljeberg, H.G.M., Akerberg, A.K.E., Bjorck, I.M. (1999). Effect of the glycemic index andcontent of indigestible carbohydrates of cereal-based breakfast meals on glucosetolerance at lunch in healthy subjects. American Journal of Clinical Nutrition 69,647–55.

Long, S.J., Sutton, J.A., Amaee, W.B., Giouvanoudi, A., Spyrou, N.M., Rogers, P.J.,Morgan, L.M. (1999). No effect of glucagon-like peptide-1 on short-term satiety andenergy intake in man. British Journal of Nutrition 81, 273–9.

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Murphy, K.G., Bloom, S.R. (2004). Gut hormones in the control of appetite. ExperimentalPhysiology 89, 507–16.

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Naslund, E., Barkeling, B., King, N., Gutniak, M., Blundell, J.E., Holst, J.J., Rossner,S., Hellstrom, P.M. (1999). Energy intake and appetite are suppressed by glucagon-likepeptide-1 (GLP-1) in obese men. International Journal of Obesity and RelatedMetabolic Disorders 23, 304–11.

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Raben, A., Tagliabue, A., Christensen, N.J., Madsen, J., Holst, J.J., Astrup, A. (1994).Resistant starch: the effect on postprandial glycemia, hormonal response and satiety.American Journal of Clinical Nutrition 60, 544–51.

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13 Metabolic Effects of Resistant Starch

Martine ChampINRA, UMR 1280, Physiologie des Adaptations Nutritionnelles, Universite deNantes, CRNH, IMAD, CHU de Nantes, Nantes, France

Resistant starch (RS) is currently defined as the sum of starch and products of

starch degradation not absorbed in the small intestine of healthy individuals

(Asp, 1992). As a consequence, RS includes not only fractions which are

resistant to endogenous enzyme digestion, but also starch and dextrins which

are potentially digestible but which, due to too short interaction with endog-

enous alpha-amylase, arrive in the colon. RS, whatever, its bioavailability, is

submitted to digestion and fermentation by colonic microbiota. Most of the RS

disappears in the colon and is metabolized into short-chain fatty acids (acetate,

propionate and butyrate) and gases (CO2, H2 and CH4).

Metabolic effects of resistant starch could a priori be classified according

to:

1. the type of metabolic effect of RS or of its metabolites; or

2. the mechanism which is involved – it can potentially be ‘direct’, due to a

matrix effect as shown for soluble (and viscous) dietary fibre, or mediated

by the short chain fatty acids produced from RS fermentation by the

colonic microbiota.

A third category is a subject of controversy. Indeed, it is linked to the effect

of the substitution of digestible starch by resistant starch. As a consequence, it

is not the metabolic effect of RS per se, but the decrease of the ‘digestible’ part

of the starch, which is mostly responsible for the metabolic effect that can be

observed.

In most investigations of metabolic studies with RS, the mechanism(s)

involved has not been elucidated fully, and/or two different mechanisms can

be simultaneously responsible for the physiological effects which have been

described. As a consequence, the first classification will be adopted all along

the review, but mechanisms involved will be discussed.

229




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In order to conclude to a metabolic effect of RS through its intrinsic

properties, the design of the study has to be faultless, particularly with regard

to the formulation of the experimental diet. Indeed, with most RS sources

containing digestible starch, the ‘true’ RS concentration in the food/ingredient

(as eaten) has to be determined prior to the clinical trial in order to adapt the

level of incorporation of the food/ingredient to this concentration.

Unfortunately, this statement is neglected in several studies, leading to a

misinterpretation of the results. Moreover, physiological or metabolic effects

can be misattributed to RS when it is present in a diet/food/ingredient which

also has a low glycemic index.

This review will mostly refer to metabolic impact of resistant starch.

Broader overviews on the physiological effects of resistant starch have been

published earlier (Champ, 2004; Champ et al., 2003).

13.1 FERMENTATION OF RS AND ITS IMPACTON COLONIC METABOLISM

RS fermentation is characterized by a significant ratio of butyrate among

SCFAs produced by its fermentation, compared to most dietary fibres (see

Tables 13.1 and 13.2).Most of the data available on RS fermentation are issued from in vitro

fermentation of RS by animal or human faecal inocula (see Table 13.1).

Studies made with effluents from ileostomy from human patients are a priori

the most accurate (Langkilde et al., 2002; Silvester et al., 1995). However an

appropriate (validated procedure) predigestion of an RS-containing food can

be considered as a relevant method to obtain the samples which have to be

submitted to in vitro fermentation (F€assler et al., 2006). Finally, many

studies have been performed on rodents or other animal species, but their

microbiota are significantly different from human microbiota and fermen-

tation profiles cannot be extrapolated to humans even if, in some cases,

a similar feature is observed between species (Wang et al., 2004; Murray

et al., 2001).

Several data of concentration of SCFAs in human stools are available in the

literature (Hylla et al., 1998; Jenkins et al., 1998; Noakes et al., 1996;

Cummings et al., 1996; Phillips et al., 1995; van Munster et al., 1994). These

are a partial reflection of fermentation, as most SCFA are absorbed in

the colon. However, they probably reflect both the intensity and the profile

of the fermentation (Table 13.2).


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Recent study from Verbeke et al. (2010) demonstrated higher concentra-

tion of butyrate in both plasma and urine from volunteers who ingested 13C

enriched intact barley kernels (containing RS and non starch polysaccharides)

compared to barley porridge (containing only non starch polysaccharides).

The investigation of RS fermentation in pigs by quantification of SCFA in

portal blood (Martin et al., 2000) raised the question of the significance of the

measurement of butyrate out of the site of production. Indeed, butyrate uptake

by the colonic mucosa seems to vary according to fermentation rate of RSwith

a higher uptakewhen fermentation is slow compared to fast fermentation. As a

consequence, quantification of butyrate in blood, urine or faeces only reflects

part of the production of butyrate.

Butyrate is the main fuel of the healthy colonocyte and is consumed by

these cells, explaining why only minor amounts of butyrate can be found in the

peripheral blood. Its depletion is thought to induce damage to the colonic

epithelium. As a consequence, dietary fibres inducing butyrate production,

such as RS, are described as ingredients that have the potential to prevent

colonic diseases such as colon cancer (Le Leu et al., 2009, 2007a, 2007b;

Clarke et al., 2008; Bauer-Marinovic et al., 2006; Perrin et al., 2001;

Wollowski et al., 2001; Hylla et al., 1998; Young & Gibson, 1995).

Butyratewould also be of interest in the healing of ulcerative colitis (Vernia

et al., 2003), even if most recent data only partly confirm earlier findings

(Hamer et al., 2010). Most studies, however, confirm that butyrate oxidation

by the colonic mucosa is impaired in various situations of inflammation (de

Preter et al., 2009; Jacobasch et al., 1999).

It is now admitted that RS can be a good substrate for butyrate production

in the colon (Le Leu et al., 2009, 2007a). The restoration of the integrity of

rat caeco-colonic mucosa in chemically induced colitis by RS, but not

by fructo-oligosaccharides (Moreau et al., 2003), probably reveals that

fermentation profile and kinetic of fermentation are determinant factors of

the benefits of dietary fibres on colonic diseases (in its broadest definition;

Howlett et al., 2010).

According to several authors, RS would improve colonic mucosal integrity

in healthy animals and/or reduce colonic and systemic immune reactivity for

which health benefits in inflammatory conditions are likely to be associated

(Nofrar�ıas et al., 2007). It would confirm the interest of RS in a prevention

context of several colonic diseases.

Apart from butyrate, RS fermentation induces production of acetate and

propionate. Both SCFAs are absorbed, but part is metabolized by the liver,

whereas mostly acetate can be found in the blood and used by peripheral

organs (Wong et al., 2006).

Metabolic Effects of Resistant Starch 231

3GCH13 08/31/2013 15:32:54 Page 232


Table 13.1 In vitro fermentation of resistant starches.

Sample Specie Total SCFA Acetate

mmol/L mmol/g mmol/L mmol/g molar

ileal subst. ileal subst. ratio

Raw banana (flour) Ileal

effluents

Human

(ileostomy)

120�2.8 5.5�0.1 62.3�3.0 2.9�0.1 55�1

vs.

Cooked banana (flour) 107�3.1 7.0�0.2 54�2.0 3.5�0.1 55�1

High RS Ileal

effluents

Human

(ileostomy)

69.6�6.9 4.9� 0.4 44.1�4.5 3.2�0.3 66�1.6

vs.

Low RS 40.9�3.9 4.8�0.3 27.9�2.5 3.3�0.2 72�1.1

mmol mmol/L molar

ratio

Raw potato starch Ileal

effluents

Pig (ileal

cannulation)

13.3 20 53.6

vs.

Control 12.3 17.5 52.3

mmol/

g OM

mmol/

g OM

Barley Native Ileal

effluents

Dog 3.61 2.66

HT extrusion (ileal

cannulation)

6.08 4.27

Corn Native 3.13 2.26

HT extrusion 3.68 3.08

Potato Native 1.64 1.46

starch LT extrusion 6.86 4.56

Rice Native 5.45 3.82


Sorghum Native 3.70 2.67


Wheat Native 2.85 2.17


mmol molar ratio

Preparation RS3 in vitro

predig. (ba)

161 31

(retrograded long chain in vitropredig. (dy)

217 35

maltodextrin)

Ileal

effluents

Human

(ileostomy)

189 34

Preparation RS2 in vitropredig. (ba)

150 32

(native high amylose

in vitropredig. (dy)

87 23corn starch)

ba: batch; dy: dynamic.

3GCH13 08/31/2013 15:32:54 Page 233

Propionate Butyrate Reference

mmol/L mmol/g molar mmol/L mmol/g molar

ileal subst. ratio ileal subst. ratio

17.0�0.5 0.8�0.0 14.0�0.5 34.8�1.1 1.6�0.1 31.0�0.9 Langkilde

et al., 2002

18.3�0.6 1.2�0.1 18 0�4 27.0�0.7 1.7�0.1 27.0�0.9

11.8�1.3 0.8�0.1 17�0.7 11.1�1.1 0.8�0.1 17�1.1 Silvester et al.,1995

6.3�0.9 0.7�0.1 16�1.1 4.5�0.6 0.5�0.1 12�0.7

mmol mmol/L molar mmol mmol/L molar

ratio ratio

6.1 8.2 22.5 4.2 5.7 15.5 Wang et al.,2004

5.9 7.8 23.2 3.2 4.5 13.5

mmol/g OM mmol/g OM

0.59 0.36 Murray et al.,2001

0.85 0.96

0.45 0.42

0 0.60

0.11 0.07

1.42 0.88

0.92 0.71

1.53 1.18

0.67 0.36

1.61 0.73

0.52 0.16

1.50 1.25

molar ratio mmol molar ratio

48 32.6 20 F€assler et al.,

2006

49 35.5 16

57 17.0 9

43 35.8 25

51 22.5 26


CH13GR 07/29/2013 16:49:36 Page 235

13.2 RESISTANT STARCH, GLYCEMIA, INSULINAEMIAAND GLUCOSE TOLERANCE

When substituting digestible starch, RS obviously reduces glycemic load of

the food/diet and, consequently, reduces insulin secretion (Raben et al., 1994).

When comparing raw potato starch (50 g containing 54% RS) to pre-

gelatinized potato starch (50 g), postprandial plasma concentrations of

glucose and insulin were lower with the RS diet, compared to the control

diet. Ranganathan et al. (1994) showed no alteration of postprandial glucose

response to a 50 g glucose load when lintnerized starch (30 g of lintner from

maize starch containing 70% amylose) was added to the experimental meal. A

lintner is a starch which has been treated with acidic solution and is poorly

fermented by the microbiota. The conclusion of the study was that the acute

effect of the ingestion of a lintnerized starch on the measured metabolic

indexes is similar to that of cellulose (insoluble and poorly fermentable fibre).

Resistant starch (RS3, PromitorTM RS, Tate & Lyle Inc, IL, USA) (from

5–25 g in a meal) has been shown to have no effect on incremental area under

the curve (iAUC) for glucose or insulin. However, there was a decrease of the

incremental blood glucose and insulin levels after a meal containing 25 g RS at

90 and 120 minutes after the meal (p¼ 0.004 and 0.001 for glucose, p¼ 0.043

and p¼ 0.042 for insulin, respectively) (Kendall et al., 2010).

The comparison of 70% amylopectin to 70% amylose corn starch (proba-

bly RS2) in normal and hyperinsulinaemic men (14-week intervention cross-

over study) revealed that the magnitude of the response in carbohydrate and

fat oxidation was blunted in hyperinsulinaemic subjects consuming excess

levels of the amylose diet (Howe et al., 1996). The hypotheses of the authors

were that this was due to:

1. an improvement in overall insulin response; or

2. a change in available substrates for oxidation resulting from microbial

fermentation.

The data from Brighenti et al. (2006) would tend to support the hypothesis

of a second-meal effect of fermentable carbohydrates such as RS. Indeed, both

a ‘slowly digestible, partly fermentable high-amylose starch (Hylon VII) plus

cellulose’ meal and an ‘amylopectin starch plus lactulose’ meal, ingested

within breakfast, improved glucose tolerance at lunch. According to these

authors, a reduction of non-esterified fatty acids (NEFA) competition for

glucose disposal might be a mechanism involved in this metabolic effect, even

if the result is not statistically significant for the RS containing meal, whereas

it is with the lactulose-containing meal (Brighenti et al., 2006).


CH13GR 07/29/2013 16:49:36 Page 236

This observation has not been made by Liljeberg et al. (1999), who

described a beneficial effect of slow absorption and digestion of starch

from a breakfast meal on glucose tolerance at the second meal (lunch),

whereas the content of indigestible carbohydrates (RS and dietary fibre) had

no effect. But the same group (Nilsson et al., 2008) demonstrated that an

evening meal containing RS and dietary fibre (barley kernel bread) signifi-

cantly improved glucose tolerance at a following standardized breakfast

(28%, compared to white wheat bread), independently of the glycemic index

of the evening meal. The benefits of RSþDF were attributed to colonic

fermentation, which may be characterized by a prebiotic effect.

de Roos et al. (1995) compared daily supplement of 30 g RS2 (high-

amylose corn starch) to RS3 (retrograded high-amylose corn starch), using

glucose as a control. They determined C-peptide excretion as a measure for

the 24-hour insulin secretion. They concluded that consumption of 30 g/day

RS3 (but not RS2) during one week reduced the insulin secretion. This result

would also suggest an impact of RS fermentation rate on insulin.

Robertson et al. (2005) suggested that dietary supplementation with RS

had the potential to improve insulin sensitivity. Indeed, insulin sensitivity

assessed by euglycaemic-hyperinsulinaemic clamp and meal tolerance test

was higher after RS supplementation (30 g RS/day during four weeks) than

after placebo treatment of healthy subjects. More recently, the same group

(Johnston et al., 2010) confirmed an improvement of insulin sensitivity of

insulin resistant subjects (40 g RS/day during 12 weeks); however, this

modification was not associated with changes in whole-body composition.

These results apparently contradict those obtained from studies on rodents.

Indeed, a high-RS diet has been shown to decrease intra-hepatic fat storage

and reduce inflammatory markers (Kabir et al., 1998; So et al., 2007).

However, these studies on animal models have compared RS to digestible

starch, which is not the case with Robertson’s studies (Robertson et al., 2005).

Kwak et al. (2012) observed that a four-week dietary treatment with rice

containing resistant starch (6.51 g RS per day, compared with control com-

posed of rice with low level of RS) reduced postprandial blood glucose and

oxidative stress, measured as plasma malondialdehyde (MDA), urinary 8-epi-

PGF (2a) (whereas RH-PAT index and total nitric oxide were increased) in

patients with prediabetes or newly diagnosed type 2 diabetes.

13.3 RS CONSUMPTION AND LIPID METABOLISM

De Deckere et al. (1993) concluded, from a study on rats fed semi-purified

diets containing a low (0.8 g/MJ) or a high (9.6 g/MJ) amount of RS, that


CH13GR 07/29/2013 16:49:36 Page 237

dietary RS can reduce serum total cholesterol and triacylglycerols concentra-

tion and fat accretion (epididymal fat pads).

Cheng& Lai (2000) observed reduced serum and hepatic cholesterol in rats

fed a diet containing 63% rice starch (richer in RS than corn starch), compared

to animals receiving corn starch as a control. The authors attributed this effect

to the significant increase of propionate in rats fed on rice starch. Propionate

may, indeed, be involved in the control of hepatic cholesterol synthesis.

However, the experimental diets had different amount of digestible starch and

it cannot be ignored that the impact on cholesterol concentrations could be

explained by the different insulin responses to the digestible starch fractions of

the diets.

Moreover, a study on germ-free rats (Sacquet et al., 1983) also demon-

strated a reduction of plasma cholesterol with RS (RS2: high-amylose starch).

In this study, the hypothesis of an effect of SCFA cannot explain the beneficial

metabolic effect. Ten years later, the same group (Math�e et al., 1993)

described a reduction of plasma cholesterol in genetically obese (fa/fa)

and lean (fa/-) rats when fed a high-amylose corn starch diet. Among the

possible mechanisms, the authors suggested an increased biliary secretion of

bile acids linked to an accelerated catabolism of lipoproteins by the liver. A

second hypothesis, which has been mentioned earlier, is the involvement on

insulin (glucagon/insulin ratio) secretion, which would explain the reduced

plasma lipid and lipoprotein levels.

The impact of RS consumption in healthy human subjects is controver-

sial. Three of the studies (Stewart et al., 2010; Heijnen et al., 1996; Behall

& Howe, 1995) failed to demonstrate any favourable effect of RS con-

sumption on lipaemia or cholesterol concentration, whereas two earlier

studies (Behall et al., 1989; Reiser et al., 1989a, 1989b) concluded to a

significant decrease of total cholesterol and triacylglycerol when the

subjects received, respectively, 247 g and 183 g per day of high-amylose

corn starch during four and five weeks (compared to high amylopectin

starch and fructose as a control). Heijnen’s 1996 study was a randomized,

single-blind, 3� 3 Latin-square study (total of 57 subjects), in which the

subjects received 30 g of raw (RS2; Hylon VII, National Starch) or

retrograded RS (RS3; Novelose, National Starch) vs. glucose during three

weeks. In the 1995 study of Behall & Howe, the subjects received

approximately 200 g raw high-amylose (vs. low-amylose) corn starch

(RS2) per day. They observed a significant increase in total cholesterol

and a decrease in triacylglycerol after the first four weeks of supplementa-

tion, whereas Achour et al. (1997) concluded from an acute study that

retrograded high-amylose corn starch (RS3) leads to a reduction in lipolysis

in the post-absorptive period.


CH13GR 07/29/2013 16:49:36 Page 238

Noakes et al. (1996) failed to observe any metabolic changes when high-

amylose starch (Hi-MaizeTM, Starch Australasia) (17 g and 25 g/day in

women and men, respectively) was introduced in the diet of hypertriglycer-

idaemic patients, whereas oat bran had a significant effect on plasma

triacylglycerol.

Results from Faisant et al. (unpublished personal data) indicated that

supplementing a normal meal with RS3 had little or no effect on postprandial

parameters of normal subjects. However, triacylglycerol metabolism was

improved, especially in subjects with higher (upper limit of the ‘normal’

range) basal triacylglycerol levels, most probably by an improvement in

clearance of postprandial chylomicron remnants.

13.4 RS CONSUMPTION, GIP, GLP-1 ANDPYY SECRETION

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are gut-secreted

peptides that have been proposed as potential anti-diabetes/obesity drugs.

These two hormones are also naturally secreted in response to meal ingestion,

but they degrade rapidly after endogenous secretion or exogenous injection.

Thus, pharmaceutical means to maintain substantial high plasma levels of

GLP-1 and PYY are intensely targeted.

RS has been reported by numerous authors to increase plasma total GLP-

1 and total PYY in rodents of both sexes at different ages (Keenan et al.,

2006; Zhou et al., 2006). For instance, Keenan et al. (2006) observed that

rats fed fermentable RS (RS2, Hi-MaizeTM 260, National Starch) had

(compared to methylcellulose) increased caecal weights and plasma PYY

and GLP-1 and increased gene transcription of PYYand proglucagon. They

conclude from their study that inclusion of RS in the diet may affect energy

balance through its effect as a fibre or a stimulation of PYY and GLP-1

expression. In the same year, Zhou et al. (2006), using in vivo and in vitro

approaches, concluded that the distal part of the gut has the ability to sense

nutrients such as butyrate, resulting in the up-regulation of PYY and

proglucagon gene expression.

Later on, the same group (Zhou et al., 2008) concluded from a study on

RS-fed rodents (Hi-MaizeTM 260 as RS2) that:

1. RS stimulated GLP-1 and PYY secretion in a substantial day-long manner,

independent of meal effect on changes in postprandial glycemia;

2. fermentation and the liberation of SCFAs in the lower gut are associated

with increased proglucagon and PYY gene expression;


CH13GR 07/29/2013 16:49:36 Page 239

3. glucose tolerance, an indicator of increased active forms of GLP-1 and

PYY, was improved in RS-fed diabetic mice.

They concluded from their study that fermentation of RS is most likely to

be the primarymechanism for increased endogenous secretions of total GLP-1

and PYY in rodents. Any factor that affects fermentation should be considered

when dietary fermentable fibre is used to stimulate GLP-1 and PYY secretion.

Tachon et al. (2013) observed that mice fed an energy-controlled diets

containing 18% or 36% type 2 RS (from high-amylose maize) were colonized

by higher levels of Bacteroidetes and Bifidobacterium, Akkermansia and

Allobaculum species in proportions that were dependent on the concentration

of the dietary fibre. The proportions of Bifidobacterium and Akkermansia

were positively correlated with mouse feeding responses, gut weight and

expression levels of proglucagon, the precursor of GLP-1.

To our knowledge, Raben et al. (1994) were the first to show that, in

humans (healthy subjects), when substituting digestible starch, RS reduces

insulin secretion but increases gastric inhibitory polypeptide (GIP), GLP-1

and epinephrine.

More recently, Nilsson et al. (2008) showed that improved tolerance was

observed after a standardized breakfast when RS and fibre (both from barley)

were ingested during the preceding evening meal. This was associated with

decreased concentration of free fatty acids and IL-6 and increased GLP-1 and

adiponectin at the time of the breakfast, thus providing evidence for a link

between the gut microbial metabolism and key factors associated with insulin

resistance.

13.5 RS CONSUMPTION, SATIETY AND SATIATIONAND FAT DEPOSITION

An acute randomised, single-blind crossover study aiming at determining the

effects of consumption of 48 g RS on appetite compared to an appropriate

control (energy and available carbohydrate-matched placebo) recently

showed a significantly lower energy intake following the RS supplement,

compared to the placebo supplement at the ad libitum test meal (Bodinham

et al., 2010). However, there was no associated effect on subjective appetite

measures.

According to de Roos et al. (1995), 30 g/day RS2 (raw high-amylose corn

starch) or RS3 (extruded and retrograded high-amylose corn starch) had little

influence on appetite and food intake. However, supplementation with RS2

caused significantly lower (p< 0.05) appetite scores than did supplementation


CH13GR 07/29/2013 16:49:36 Page 240

with RS3 or glucose, though subjects paradoxically felt less full while

consuming RS2. This feeling might be related to a higher hydration capacity

of extruded retrograded starch compared to raw starch.

Kendall et al. (2010) compared the impact of lower doses of RS3 (from

5–25 g of PromitorTM RS, Tate & Lyle Inc, IL, USA) in a meal. Feelings of

fullness were greater with the 5 g dose of RS compared to control, while the

satiety quotient for overall appetite was significantly greater for 25 g RS in the

early phase after the eating period (p¼ 0.036, 0.075 and 0.97 at 45, 30 and

15 minutes after lunch).

In several studies, RS seems to have an impact on satiety which could be

mediated by acetate produced during colonic fermentation. Indeed, RS

reduced subjective satiety ratings in the intermediate absorptive period (Raben

et al., 1994; Achour et al., 1997) but led, in the post-absorptive period, to

higher subjective scores of satiety, which may reduce subsequent food

intake during the next meal (Achour et al., 1997). The lower subjective

scores for satiety and fullness after the RS (RS2; raw potato starch) than

after digestible starch (pregelatinized potato starch) were associated with

lower postprandial plasma concentration of glucose, insulin, GIP and GLP-1

(Raben et al., 1994).

Nilsson et al. (2008) observed the effect of an evening meal containing RS

(high-amylose barley kernel) or b-glucan (b-glucan-rich barley kernel) on

satiety after a subsequent standardized breakfast (ten hours after the evening

meal). Satiety was positively correlated to breath hydrogen, an indicator of

colonic fermentation.

Monsivais et al. (2010) recently showed that soluble fibre-dextrin (SFD)

enhanced the satiating power of beverages, compared to soluble corn fibre,

polydextrose and resistant starch. Indeed, only soluble fibre dextrin signifi-

cantly suppressed energy intake (p¼ 0.023). This SFD could be considered as

part of RS3. The SFD is probably highly fermentable, and its satiating effect

might also be mediated by SCFA produced within the four hours following the

fibre intake. Similar results on resistant dextrins were obtained by Nazare

et al. (2011).

Anderson et al. (2010) concluded from their recent study that estimates of

starch digestibility by the in vitro Englyst method were able to predict food

intake in young men 30 minutes and 120 minutes after consumption of a soup

containing 50 g maltodextrin, whole grain, high-amylose, regular cornstarch

or no added starch.

As shown by Tagliabue et al. (1995), RS (potato starch, in this study) seems

to have no thermogenic effect and its presence does not influence the size of

the thermic response to digestible starch. RS intake was, as expected followed

by lower oxidation and greater fat oxidation.


CH13GR 07/29/2013 16:49:36 Page 241

Lerer-Metzger et al. (1996) concluded that the replacement of 570 g wheat

starch/kg diet with mung-bean starch for five weeks resulted in a reduction in

plasma triacylglycerol concentration and adipocyte volume in both normal

and diabetic rats. Thus, the type of starch mixed into the diet may have

important metabolic consequences in normal and diabetic rats.

Robertson et al. (2005) showed that a supplementation with RS lead to

increased insulin sensitivity and a reduction in glycerol and free fatty acids

across subcutaneous adipose tissue. This was suggestive of a change in

adipocyte metabolism that may lead to a change in insulin sensitivity. RS

and inulin have been demonstrated to increase the release of gut hormones

with roles in appetite regulation and possibly, leptin release (Delzenne et al.,

2005; Keenan et al., 2006).

Keenan et al. (2006) and Zhou et al. (2006) have shown that dietary

resistant starch increased GLP-1 and PYY secretion. The same group (Zhou

et al., 2008) found that:

1. RS stimulated GLP-1 and PYY secretion in a substantial day-long manner,

independent of meal effect on changes in postprandial glycemia;

2. fermentation and the liberation of SCFAs in the lower gut are associated

with increased proglucagon and PYY gene expression;

3. glucose tolerance, an indicator of increased active forms of GLP-1 and

PYY was improved in RS-fed diabetic mice.

They concluded from their study that fermentation of RS is most likely the

primary mechanism for increased endogenous secretions of total GLP-1 and

PYY in rodents.

In an acute, randomizeddouble-blind, cross-over study comparing the effects

of four fibre and a low-fibre treatment on satiety, Willis et al. (2009) observed

that resistant starch (Novelose 330 and Hi-MaizeTM 260, National Starch;

RS3 and RS2, respectively) and corn bran had the most impact on satiety,

whereas polydextrose had little effect and behaved like the low-fibre treatment.

The same group (Willis et al., 2010) showed that satiety, gut hormone

(ghrelin, GLP-1, and PYY (3–36)) response, and food intake at subsequent

meals did not change in a dose-dependent manner after subjects consumed 0,

4, 8 and 12 g of mixed fibre in muffins for breakfast. However, despite lack of

differences in satiety, gut hormone levels differed among treatments. Ghrelin

was higher after the 12 g fibre dose than after the 4 g and 8 g fibre doses; GLP-

1 was higher after the 0 g fibre dose than after the 12 g and 4 g fibre doses; and

PYY(3–36) did not differ among fibre doses.

According to Shen et al. (2009), the mechanism of decreased body fat by

RS would be linked to increased neuropeptide POMC gene expression in the


CH13GR 07/29/2013 16:49:36 Page 242

hypothalamus, and such an effect is independent of the involvement of

visceral afferent capsaicin-sensitive neurons.

So et al. (2007) investigated the impact of RS on body fat patterning and

central appetite regulation in mice. Mice receiving high RS (HRS; high-

amylose corn starch (60% RS)) diet during eight weeks had similar body

weights than those fed the low RS (LRS) diet, although their total body

adiposity, subcutaneous and visceral fat, intrahepatocellular lipids, plasma

leptin, plasma adiponectin and plasma insulin/glucose ratios were signifi-

cantly lower than the low RS group. Adipocytes isolated from the HRS group

were significantly smaller and had higher insulin-stimulated glucose uptake.

Manganese-enhanced magnetic resonance imaging (MEMRI) of the ventro-

medial and paraventromedial and paraventricular hypothalamic nuclei

suggested a satiating effect of the HRS diet despite a lower energy intake.

These data suggest that there may be appetite regulatory differences between

animals exposed to varying amount of RS, with the LRS animals producing

MEMRI data closer to fasted animals, and the HRS animal producingMEMRI

data closer to that of satiated animals.

So et al. (2007) suggest a link between short chain fatty acids production

from the fermentation of RS and the change in adipocyte morphology. Indeed,

the G protein receptor (GPCR43) on adipocytes may play a role in adipocyte

differentiation and proliferation; the ligands for these receptors appear to

be the short-chain fatty acids, acetate and propionate (Brown et al., 2003).

There is recent evidence that activation of GPCR43 by acetate and propionate

causes a decrease in leptin secretion and an increase in adipocyte differentia-

tion (Hong et al., 2005).

Significantly larger adipocyte size and over-insulin-stimulated glucose

oxidation in rats fed diets low in RS, compared with rats fed high RS diets,

had been described earlier by Kabir et al. (1998). So et al. (2007) also noticed

a significant difference in plasma leptin concentrations, with the low RS group

having significantly higher levels despite similar body weights. This would

confirm the observation made earlier by Skurk et al. (2007) that leptin levels

are driven by the lipid content and size of the adipocyte rather than by the

overall body weight. The concomitant increase in adiponectin and leptin in the

mice with low RS diet, compared to ‘high RS’ mice, is consistent with an

induction of leptin resistance in these mice (So et al., 2007).

13.6 CONCLUSION

Resistant starches are not homogeneous entities, as they can be totally or

partly fermented in the colon and their fermentability rate differs from one to


CH13GR 07/29/2013 16:49:36 Page 243

another. These various properties probably explain some of the contradictory

results described in the literature. Their fermentation produces a significant

amount of butyrate but, depending on the methodology used, butyrate or

propionate can be the second SCFA produced during fermentation of RS, after

acetate. Butyrate, being the nutrient of the colonocyte, is usually taken up

by the colonic mucosa, and only minor amounts can be found in the plasma

(or in urine).

When digestible starch is substituted in the diet by RS, glycemic load

and, consequently, insulin secretion is reduced. In the long term, RS

seems to be able to improve insulin response in hyperinsulinaemic

subjects. The impact of RS consumption at dinner on a subsequent

breakfast revealed the involvement of RS fermentation on improvement

of glucose tolerance and insulin sensitivity in healthy and insulin-

resistant subjects.

GLP-1 and PYY, two gut-secreted peptides which have been proposed as

potential anti-diabetes/obesity drugs, are also naturally secreted in response to

meal ingestion. RS has been reported to increase:

1. plasma total GLP-1 and total PYY in rodents; and

2. gene expression of PYY and proglucagon, probably through its

fermentation.

GLP-1 has been shown to increase in healthy subjects when part of the

digestible starch was substituted by RS. This impact of RS on gut peptides has

been confirmed within studies, showing an improved tolerance observed after

a standardized breakfast when RS (and fibre) was ingested during the

preceding dinner. It is associated to increased GLP-1 and adiponectin at

the time of the breakfast. These findings provide evidence for a link between

RS fermentation and the beneficial impact on insulin resistance. GLP-1

secretion would also explain the satiating effect of RS at several hours after

its ingestion.

Recent works have suggested a change in adipocyte metabolism that may

also lead to a change in insulin sensitivity. A link between short-chain fatty

acids production from the fermentation of RS and smaller adipocytes,

associated with better prognostic in metabolic syndrome, also supports the

beneficial metabolic impact of some RS. There is, however, still much work to

be performed in order to obtain new claims on several of the resistant starches.

Indeed, the effects will have to be demonstrated on each type of RS, unless a

clear correlation can be made between each fermentation pattern and corre-

sponding metabolic effects.


CH13GR 07/29/2013 16:49:36 Page 244

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Wollowski, I., Rechkemmer, G., Pool-Zobel, B.L. (2001). Protective role of probioticsand prebiotics in colon cancer. American Journal of Clinical Nutrition 73(2 Suppl),451S–455S.

Wong, J.M., de Souza, R., Kendall, C.W., Emam, A., Jenkins, D.J. (2006). Colonic health:fermentation and short chain fatty acids. Journal of Clinical Gastroenterology 40(3),235–43.

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14 The Microbiology of Resistant StarchFermentation in the Human LargeIntestine: A Host of UnansweredQuestions

Harry J. FlintMicrobial Ecology Group, Rowett Institute of Nutrition and Health,University of Aberdeen, Aberdeen, UK

14.1 INTRODUCTION

The human intestinal tract carries a large and diverse community of resident

microorganisms. The highest cell densities (1011 per gram) are found in the

large intestine, where flow rates are slowest and conditions in the gut lumen

become anaerobic. Some energy for microbial growth is derived from

endogenous sources through secreted proteins such as mucin and digestive

enzymes, and sloughed epithelial cells. In general, however, the main energy

sources are carbohydrates that have remained undigested in the stomach and

the small intestine (Macfarlane & Gibson, 1997).

Many plant oligosaccharides and cell wall polysaccharides are undegrad-

able, or poorly degraded, by mammalian digestive enzymes. Perhaps surpris-

ingly, however, for many diets it is a fraction of the potentially digestible

polysaccharide starch (‘resistant starch’) that escapes small intestinal diges-

tion which is estimated to provide the single largest energy source for

microbial growth in the colon (Cummings et al., 1996). There is increasing

evidence that the fermentation of resistant starch in the human large intestine

may lead to health benefits that include prevention of colorectal cancer and

improved insulin responses (Hylla et al., 1998; Bird et al., 2000, 2008;

Robertson et al., 2005).

Despite extensive early cultural studies on polysaccharide utilization by

human colonic bacteria (Salyers et al., 1977a, 1977b), our understanding of

the microbiology of resistant starch fermentation is still rather limited. One

251




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problem is that it is not straightforward to reproduce, under in vitro conditions,

the state of the undigested starch particles that arrive in the large intestine

(Annison & Topping, 1994). Microbiological studies on pure cultures neces-

sarily tend to use autoclaved starch preparations, whose properties may differ

from those of starch in small intestinal effluent (Englyst &Macfarlane, 1986).

A second problem lies with the microbial community, which, in the colon,

is dominated by anaerobic bacteria that are not easily cultivated. Despite a

rapid increase recently in sequence-based diversity studies, relatively few

laboratories work routinely with anaerobes, making functional information

scarce. There are, however, excellent prospects now for combining molecular

and cultural approaches to obtain new insights into the impact of resistant

starches on microbial populations in the human colon.

14.2 IDENTIFYING THE MAJOR DEGRADERS OFRESISTANT STARCH IN THE HUMANGI TRACT

14.2.1 The human colonic microbiota

Direct sequence analysis of DNA and RNA from faecal and gut samples has

revealed a more complete picture of the diversity of bacteria that colonize the

human large intestine than was possible using culture-based approaches. Most

studies agree that only one-third of the bacterial diversity estimated from

16S rRNA gene sequencing is currently represented by named, cultured

species (Suau et al., 1999; Hold et al., 2002; Eckburg et al., 2005). On

the other hand, a much higher percentage of the numerically predominant

species are available as cultured isolates, so that more than half of amplified

ribosomal sequences detected in faecal samples can correspond to cultured

species (Walker et al., 2011).

The two most abundant bacterial phyla are the gram-positive Firmicutes

and gram-negative Bacteroidetes, followed by the Actinobacteria and

Proteobacteria, with small numbers of other phyla such as Verrucomicrobia.

The molecular diversity of eukaryotic gut microbes (protozoa and fungi) has

received comparatively little study to date.

14.2.2 Cultural studies

Extensive early bacterial isolation work using anaerobic techniques revealed a

large number of human gut bacteria that are capable of growth on starch.

Salyers et al. (1977a, 1977b) reported finding starch utilizers within the

Bacteroidetes, Firmicutes and Actinobacteria, with this activity being


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particularly common among isolates of Bacteroides spp. (Table 14.1).

Subsequent selective isolations on media using starch as the sole energy

source produced a high proportion of Bifidobacterium spp. (Actinobacteria)

(Macfarlane & Englyst, 1986). The number of starch-degrading species from

these early studies among what is normally the most abundant phylum, the

Firmicutes, remained low, but it now appears that this may reflect the

relatively low coverage of this phylum previously by cultured strains (Eckburg

et al., 2005).

14.2.3 16S rRNA-based studies

While 16S rRNA-based community analysis reveals the diversity of human

colonic bacteria, it does not by itself provide any functional information. On

the other hand, when used in conjunction with selective functional screens, it

can potentially reveal uncultured starch-degrading species. Leitch et al.

(2007) used 16S rRNA sequencing to study the colonization of insoluble

resistant starch (Hylon VII) by mixed human faecal bacteria in an anaerobic

fermentor system, and 80% of starch-attached sequences recovered from four

different microbial communities corresponded to four species – Eubacterium

rectale, Ruminococcus bromii, Bifidobacterium adolescentis and Bifidobac-

terium breve (Figure 14.1).

This represents a very small subset of the diversity present in the starting

inocula, showing that the ability to bind to and colonize resistant starch is quite

limited among human colonic bacteria. The fact that the predominant attached

bacteria belong to cultured species suggests that the major colonizers may

already have been identified from the earlier isolation studies.

Another elegant functional approach is provided by stable isotope probing.

Kovatcheva-Datchary et al. (2009) incubated 13C labelled resistant starch

with mixed human faecal bacteria and recovered 13C labelled nucleic

acid, resulting from starch utilization, by density gradient centrifugation.

Table 14.1 Amylolytic activity in human gut anaerobes.

Growth with:

Strains tested amylose amylopectin

Bacteroides spp. 188 72% 73%High GþC Actinobacteria 52 31% 52%Low GþC Firmicutes 38 16% 53%

Data from Salyers et al. (1977a, 1977b).

The Microbiology of Resistant Starch Fermentation in the Human Large Intestine 253

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16S rRNAanalysis again revealed that themajor utilizerswere bifidobacteria,

R. bromii andE. rectale. The last twoare low%GþCbacteria belonging to the

Firmicutes, emphasizing that members of this phylum also play an important

role in starch utilization. This is also apparent from analysis of the responses

among the faecal microbiota to added dietary starch in vivo, discussed

further below.

14.3 SYSTEMS FOR STARCH UTILIZATIONIN GUT BACTERIA

The availability of genome sequence data for an increasing number of starch-

utilizing bacteria opens up new prospects for analyzing the amylolytic gene

complement of starch-degrading species. Interpretation of these sequence

data, however, depends critically on the relatively small number of detailed

Figure 14.1 Colonization of insoluble substrates by human faecal bacteria. Mixedfaecal bacteria were introduced into a porous bag containing insoluble substrate(Hylon VII starch, wheat bran or porcine mucin) within an anaerobic continuous flowfermentor system. The experiment was repeated with inocula from four differenthealthy individuals. The predominant bacteria attached to each type of substratewere estimated by amplification and sequencing of 16S rRNA genes, as described byLeitch et al. (2007).


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functional investigations that have been carried out in cultured gut bacteria

(Flint et al., 2008).

14.3.1 Bacteroides spp.

The starch utilization system of B. thetaiotaomicron is better understood than

that of any other human gut bacterium, thanks to the detailed studies of Salyers

et al. (reviewed Xu & Gordon, 2003; Flint et al., 2008). This gram-negative

species has a double cell membrane enclosing a periplasmic space. Soluble

starch molecules are proposed to be trapped by a complex of Sus proteins

located on the outer membrane surface. The molecules are then subject to

limited hydrolysis and translocation into the periplasmic space, where the

majority of the amylolytic activity is located. The major amylases are

described as neopullulanases, with the ability to hydrolyse a(1–4) linkagesin amylopectin and pullulan as well as in amylose.

14.3.2 Bifidobacterium spp.

Several species of bifidobacteria found in the human colon – mainly B. breve,

B adolescentis and B. pseudocatenulatum – show amylolytic activity, and

these include isolates with activity against high-amylose starches (Wang et al.,

1999; Ryan et al., 2006). A major cell surface amylase was identified in

B. breve that contains separate pullulanase and amylase catalytic domains,

together with a C terminal sortase cell wall anchoring signal (Figure 14.2).

The pullulanase domain was shown to debranch amylopectin by cleaving

Figure 14.2 Domain structures of the major cell surface amylases from two speciesof Gram-positive bacteria found in the human large intestine. GH13 – glycosidehydrolase family 13 catalytic domain; CBM – carbohydrate bindingmodule. Adaptedfrom Flint et al. (2012) Gut Microbes 3:4, 289-306. Fig. 3A.


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a(1–6) linkages and acts in synergy with the a-amylase that cleaves a(1–4)linkages. Disruption of this gene in B. breve eliminated the ability to grow on

starch (Motherway et al., 2008).

14.3.3 Lachnospiraceae - Roseburia spp., Eubacteriumrectale and relatives

Bacteria related to Roseburia spp. and Eubacterium rectale represent an

important group of butyrate-producers in the human colon, accounting for

around 7–10% of bacteria in human faeces (Aminov et al., 2006). Most

isolates from this group can utilize starch as a growth substrate (Ramsay

et al., 2006). R. inulinivorans A2-194 produces a prominent amylase that is

bound to the cell surface via a sortase-mediated step. This multi-domain

enzyme includes putative starch binding modules, together with an a(1, 4)amylase (neopullulanase) catalytic domain that acts on pullulan and amylose.

The R. inulinivorans amylase, therefore, has many similarities to the enzyme

described from B. breve (Figure 14.2). These features appear consistent with

extracellular hydrolysis of starch molecules combined with the uptake and

processing of malto-oligosaccharides, as in B. breve.

14.3.4 Ruminococcaceae

This family of Firmicutes bacteria is numerically abundant in the human

colon, but is quite poorly represented by cultured strains. Ruminococcus

bromii has been regarded as a specialist starch-degrading species, but little if

any work appears to have been done on its amylase system. Recent work,

however, showed that the enzyme system of R. bromii was more effective

in degrading several particulate resistant starches than the amylases of

B. thetaiotaomicron, E. rectale or B. adolescentis (Ze et al., 2012). This

difference was most apparent when these starches were incubated without

heating, or after boiling for ten minutes, whereas autoclaving increased

degradability by the other species. Relatives of R. bromii were previously

shown to be preferentially associated with particulate material in human

faecal samples (Walker et al., 2008).

14.4 METAGENOMICS

Extraordinary recent advances in rapid sequencing capability are permitting

analysis of the gene complement of complex microbial communities (e.g.

Kurokawa et al., 2007). This allows the frequency of amylase genes, for


3GCH14 07/29/2013 17:4:16 Page 257

example, to be monitored in the community in response to dietary change.

This approach should be seen as complementing, rather than replacing, the

need for organism-based studies. First, the annotation of genomic data

depends crucially on functional information, which is still limited. Second,

as discussed above, the possession of an amylase gene does not define the role

of an organism in starch utilization, which is determined by the complete

enzyme complement and the organization of the relevant catalytic activities

and transport capabilities.

14.5 FACTORS INFLUENCING COMPETITION FORSTARCH AS A GROWTH SUBSTRATE

It is expected that the chemical and physical structure of resistant starch

entering the large intestine will influence the types of micro-organism capable

of exploiting it.While it seems likely that the different types of enzyme system

described above are equipped to deal with different forms of starch, there is as

yet little experimental evidence to support this notion. The failure to detect

Bacteroides among bacteria attached to resistant starch particles in vitro

(Leitch et al., 2007) perhaps suggests that Bacteroides are better able to utilize

solubilized than particulate starches, but this possibility needs more rigorous

testing. Several studies suggest that bifidobacteria are particularly active

against high-amylose starches (Wang et al., 1999) but, again, direct compari-

sons with amylolytic Firmicutes bacteria do not appear to have been made.

The gut environment can also play a role in determining the types of

bacteria that utilize a given form of starch, and therefore which bacterial

populations may be stimulated by the addition of resistant starch to the diet. A

good example is the influence of gut pH. Active microbial fermentation in the

proximal colon results in mildly acidic conditions, whereas pH is closer to

neutrality when fermentation is less active, e.g. in the distal colon.

In a fermentor system supplied continuously with soluble starch and other

polysaccharides at pH 5.5, the microbial community comprised approxi-

mately 25% of E. rectale-related butyrate-producing bacteria and 25%

Bacteroides relatives. On switching to pH 6.5, however, Bacteroides spp.

became dominant (80%) andE. rectale relatives became undetectable (Walker

et al., 2005; Duncan et al., 2009).

Since Bacteroides spp. appear to be less tolerant of growth inhibition by

weak acids at reduced pH than many gram-positive species, it appears that the

mildly acidic pH creates the opportunity for the gram-positive species to

compete successfully with Bacteroides for the polysaccharide substrates.

The consequence of these differences in pH tolerance, via their effects on


3GCH14 07/29/2013 17:4:16 Page 258

the balance of the microbial community, was a four-fold higher butyrate

concentration in the fermentor at pH 5.5, compared to that at pH 6.5 (Walker

et al., 2005).

14.6 METABOLITE CROSS-FEEDING

The anaerobic microbial communities of the rumen and large intestine are

characterized by high cell densities and by extensive interspecies cross-

feeding of fermentation products (Flint et al., 2007). Addition of resistant

starch to the diet could, therefore, lead potentially to the indirect stimulation of

many groups of bacteria and their metabolic products, in addition to the

primary amylolytic species. B. adolescentis L2-32, for example, degrades

starch to produce lactate, acetate and formate. E. hallii cannot degrade starch,

but it can convert acetate and lactate into butyrate. Co-cultures of these two

bacteria convert starch largely into butyrate, with growth of both B. adoles-

centis and E. hallii (Duncan et al., 2004; Belenguer et al., 2006). Cross-

feeding of partial degradation products from polysaccharide substrates is also

a well established phenomenon (Belenguer et al., 2006; Falony et al., 2006;

Figure 14.3).

Starch

Bifidobacterium spp.

Oligosaccharides

acetateL-lactateformate

Eubacterium hallii

12

fermentation (1 –

Roseburia hominisproducts

substrate 2 –“spillover”) butyrate

Figure 14.3 Potential contribution of metabolite cross-feeding to butyrate formationfrom resistant starches. Two distinct mechanisms are shown, one involving cross-feeding of fermentation products (lactate and acetate) and the other cross-feeding ofpartial breakdown products from starch. E. hallii is a lactate-utilizing bacterium thatproduces butyrate, while R. hominis is a non-lactate utilizing butyrate producer;neither species can grow by itself on resistant starch, but both grew when partneredwith a starch-degrading Bifidobacterium adolescentis strain. Based on Belengueret al. (2006).


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14.7 IMPACT OF DIETARY RESISTANT STARCH UPONCOLONIC BACTERIA AND BACTERIALMETABOLITES IN HUMANS

The application of molecular approaches to bacterial enumeration in human

dietary intervention studies can reveal microbial responses to resistant starch

(RS) in vivo. Almost invariably, these responses have to be monitored in stool

samples. These can be expected to provide a good reflection of the microbiota

of the colonic lumen, but the temporal relationship between changes in the

proximal colon and changes seen in stool samples, of course, depends on

transit time.

Abell et al. (2008), using 16S rRNA based DGGE profiling, detected an

increase in relatives of R. bromii following increased RS intake. Another

recent study looked at the impact of diets high in NSP (wheat bran) or high in

type III resistant corn starch, each consumed for three weeks by 14 overweight

males, using a cross-over design (Walker et al., 2011). Bacterial groups were

monitored by qPCR, and for six subjects by 16S rRNA sequencing. This

analysis detected rapid increases in R. bromii-related bacteria and in the

Eubacterium rectale/Roseburia groups on the RS diet, although responses

varied markedly between individuals. Perhaps surprisingly, only one individ-

ual showed a strong response for bifidobacteria. A previous study failed to

detect a response in Eubacterium-related bacteria in humans consuming diets

high in RS (Schwiertz et al., 2002), but E. rectale was not monitored.

Physiological effects of RS intake are also evident from human studies, in

particular changes in faecal SCFA and improvements in insulin resistance

(Robertson et al., 2005). Evidence for protection against colorectal cancer

comes largely from animal studies and is proposed to occur mainly through

the butyrogenic effect of resistant starch (McIntyre et al., 1993). Among the

amylolytic groups known to be abundant in the human colon, only relatives of

E. rectale are butyrate-producers. As mentioned above, however, metabolite

cross-feeding might account for a butyrogenic effect of RS, even when the

dominant bacteria stimulated by starch are not butyrate-producers. Changes in

SCFA production rates have the potential to alter physiological functions

including appetite, inflammation and gut motility through signalling to gut

receptors (Brown et al., 2003).

The delivery of additional fermentable carbohydrates to the colon can have

numerous other consequences. Depression of colonic pH has been already

discussed above, and this is known to influence Ca2þ availability as well as

microbial competition. Another important effect is the increased incorpora-

tion of nitrogen into microbial cell protein, resulting from increased bacterial


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growth and biomass, since this tends to decrease the concentration of

nitrogenous compounds available for catabolism to toxic or carcinogenic

products in the colon (Gill & Rowland, 2002).

14.8 CONCLUSIONS AND FUTURE PROSPECTS

Animal studies have shown that different types of RS can differ substantially

in their effects on gut metabolism and host physiology (Le Leu et al., 2009).

Such variation can be assumed also to occur in humans. At the same time,

there is evidence that RS fermentation may vary markedly between indi-

vidual humans (Walker et al., 2011). Host factors, the nature of the dietary

substrate and the composition of the gut microbiota are all likely to play a

role in determining the fermentability of resistant starch in the large

intestine (Figure 14.4).

The likely inter-dependence of these various factors is explored briefly

below.

1. RS structure will determine its rate and site of fermentation in the large

intestine. The structure of dietary starch (particle structure, association

with other polymers, crystallinity, branching, retrogradation, cross-linking)

Dietary starchDietary factors –

starch structure, particle size

cooking, storage

host amylases

digestion

(upper GI tract)

amylase inhibitors Host factors –

secretions

turnover

Resistant starch

gut environment

microbial amylases

Gut microbiota –

species

compositionfermentation

(large intestine)

Figure 14.4 Factors influencing the fate of starch in the intestine.


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is known to determine its digestibility in the small intestine. It can be

assumed that these characteristics will also determine how rapidly resistant

starch is fermented in the colon, but less research has been done on this.

Rapid fermentation ofRSmay lead to complete fermentation in the proximal

colon, whereas slower rates will also support fermentation in more distal

regions, with the possibility of incomplete colonic fermentation overall.

2. Different types of RS may promote different groups of colonic bacteria. It

is possible that different forms of RS will prove to be accessible by

different groups of colonic microorganisms. This could result in selective

effects of starch intake upon the species composition of the colonic

microbiota, as well as differential effects on gut metabolism. For example,

it was suggested above that Bacteroides spp. may be best adapted to

utilizing soluble starch molecules. It has also been proposed that certain

types of RS might be more bifidogenic because of the high activity of this

group on high-amylose starches. If such selective effects occur, then

individuals who differ in the composition of their gut microbiota can

be expected to show different responses to RS intake as, indeed, is

suggested by the recent work of Martinez et al. (2010).

3. Host and dietary factors can influence what fraction of starch comprises

RS. Host factors (e.g. affecting secretion of digestive enzymes or transit

times) could play an important role in determining the undigested starch

fraction that arrives in the large intestine and its subsequent rate of

fermentation in the colon. This is also likely to be true for dietary factors

(e.g. the pattern of starch ingestion over time) and the presence of amylase

inhibitors of dietary origin (Wolin et al., 1999). Thus, it may be that the

types of starch that comprise RS will differ between individuals and also

within individuals over time, driven by changes in dietary intake.

4. Multiple factors (host genotype, diet, health, medication) have the poten-

tial to alter the amylolytic community within the large intestine. It seems

possible that a few species of amylolytic anaerobe play key roles in the

initial degradation of RS in the colon, especially when the RS exists in

particulate form. If so, variation in these ‘keystone’ species between

individuals could result in individual differences in fermentability of

RS (Ze et al., 2012). One possibility is that certain species might be

eliminated by antibiotic treatment during an individual’s lifetime and may

fail to re-establish, as has been proposed, for example, to explain variations

in oxalate degradation between humans (Duncan et al., 2002).

We have to conclude that some of the most fundamental questions about

the factors that influence starch fermentation in the human colon, and its

consequences for human health, remain unanswered. A combination of


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cultural microbiology, microbial genomics and molecular ecology promises

progress in understanding microbial fermentation of starch by colonic micro-

organisms. This effort will clearly need to be allied, however, to an under-

standing of starch chemistry, and to physiological and nutritional studies both

in humans and in animal models.

ACKNOWLEDGEMENTS

The author would like to acknowledge support from the Scottish Government

Rural and Environment Research and Analysis Directorate.

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15 Colon Health and Resistant Starch:Human Studies and Animal Models

Suzanne Hendrich, Diane F. Birt, Li Liand Yinsheng ZhaoInterdepartmental Graduate Program in Genetics, Department of FoodScience and Human Nutrition, Nutrition and Wellness Research Center,Iowa State University, USA

15.1 RS CLASSIFICATION

Resistant starches (RS) are named for their resistance to digestion in the small

intestine. This resistance allows them to pass into the large intestine and serve

as a substrate for microbial fermentation. RS have been categorized into four

types (Haralampu, 2000). Type 1 RS is physically inaccessible to digestive

enzymes such as starch from coarse grains. RS type 2 is ungelatinized starch,

including starch from potatoes or high-amylose corn starch, with long chains

that are uniformly packed (Jane, 2006). RS type 3 is starch that has been

cooked, then cooled, and is retrograded (Haralampu, 2000). It is the most

thermally stable RS and is exemplified by potato starch in cold potato salad.

RS4 is chemically modified starch that is, for example, cross-linked in a

manner to restrict swelling of the starch granules (Jane, 2006).

15.2 RS AND COLON HEALTH: OVERVIEW

Colon health involves maintenance of normal function of the colon and the

prevention of colon diseases. Healthy colon function permits regular bowel

movement on a daily basis or somewhat more frequently, consisting of

relatively soft but non-diarrhetic faeces. Colon health may be reflected in

the measurement of laxation (the frequency and total amount of faeces

excreted) and in the avoidance of constipation, which may be defined as

bowel movement frequency less than three times per week (Marks, 2009).

267




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There is little data to support what daily faecal weight should be considered to

be healthy, but one study of 53 Australians showed that the 16 subjects with

daily faecal mass of >150 g had higher faecal butyrate concentrations and

lower ammonia concentrations in faeces, which were thought to be protective

against colon cancer (Birkett et al., 1997).

Colon diseases include colon cancer, irritable bowel syndrome (IBS – also

referred to as spastic colon), inflammatory bowel diseases or colitis (e.g.

Crohn’s disease, ulcerative colitis), and diverticulosis/diverticulitis. The roles

of RS in preventing or protecting from each of these conditions are not yet

well understood, but seem to be most closely related to improved laxation and

increased short chain fatty acid (SCFA) production. The ability of RS to shift

gut microbial populations to increase probiotics (i.e. beneficial microbes)

and/or to decrease detrimental or pathogenic microbial species may also play

a role in disease protection. The biology and chemistry of interactions

between RS and the gut has been characterized to at least some extent in

varying models. This work provides a basis for further investigation of RS in

colon health and disease.

15.3 RS, GUT MICROBES ANDMICROBIALFERMENTATION

Several innovative model systems have enriched the understanding of how RS

affects gut microbial populations and their metabolic products. Germ-free

F344/N rats were orally dosed with pooled human faeces from three indi-

viduals from the UK or from Italy and were fed either a high sucrose diet or

15% RS (CrystaLeanTM) substituted for a portion of the sucrose for four

weeks (Silvi et al., 1999). Body weights of male rats fed RS were significantly

greater than for sucrose-fed males, whereas body weights of female rats fed

RS and bearing microbes from individuals from the UKwere significantly less

than in sucrose-fed females. Counts of Lactobacilli and Bifidobacteria were

increased, and Enterobacteria were decreased by RS feeding compared with

sucrose feeding. Staphylococci and Streptococci were increased by RS in rats

bearing microflora from humans from the UK, whereas Staphylococci were

decreased in rats fed RS and bearing Italian human microbes. Cecal b-gluco-sidase activity was increased and ammonia concentration was decreased by

RS. Cecal b-glucuronidasewas only decreased by RS in rats bearing microbes

from humans from the UK. Cecal butyrate was increased and propionate was

decreased by RS feeding (Silvi et al., 1999). Butyrate enhanced tight

junctions, improving the epithelial barrier function of Caco-2 cells, a widely

used model for human intestinal uptake and metabolism (Peng et al., 2009).


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This barrier function is thought to be important for protection from colon

diseases.

As reviewed by Wollowski et al. (2001), the above changes in human

microflora-associated rats by RS to increase lactic acid bacteria (LAB;

Lactobacilli and Bifidobacteria) may help to detoxify some carcinogens, lower

faecal pH which is associated with colon cancer prevention, and stimulate

butyrate production, which improves protection of colonocytes from oxidative

stress, and maintains normal colon cell function. Note that LAB produce

lactate, which is then a substrate for butyrate-forming species (such as

Clostridia cluster XIVa microbes) (Collins et al., 1994), so the interactions

among gut bacteria needed for healthy colonic metabolism are complex.

Additional studies of RS feeding to female BALB/c mice showed that

increasing high-amylose corn starch intake from 0–30 (or 40% of the diet)

increased Bifidobacteria counts after 25 days (Wang et al., 2002). When

Bifidobacterium Lafti 8B was co-administered orally, the 40% high-amylose

starch diet also increased faecal butyrate concentrations by about fourfold

compared to control starch feeding (Wang et al., 2002), lending further

support to the concept that RS may benefit both gut bacterial composition

and microbial metabolism.

In vitro anaerobic incubations of resistant starches or their residues (starch

remaining after a-amylase treatment to simulate human starch digestion) from

various sources with human faeces also provide models for examining gut

microbes andmicrobialmetabolism.Pyrodextrin residues (heat-treated starches

creating RS type 4) from potato, lentil and cocoyam increased the propionate:

acetate ratio, but did not alter butyrate in human faecal incubations, compared

with residues from the non-resistant parent starches (Laurentin & Edwards,

2004).AnRS type 3 polymorphmade from thermal processing of high-amylose

cornstarch (HACS) increased butyrate production and Bifidobacteria counts in

continuous batch fermentations with human faecal inocula over 11 days,

compared with the control HACS (Lesmes et al., 2008). Thus, a human faecal

incubationmodel produces generally similar results to rodentmodels, regarding

the ability of RS to increase butyrate and Bifidobacteria, two factors thought to

benefit colon health. Proof of such benefits in human feeding studies remains to

be determined. A few relevant and recent studies regarding some aspects of

colon health are further described in the following.

15.3.1 RS and laxation

Because RS provides indigestible carbohydrates to the colon, RS should be

considered to be a subclass of dietary fibre. RS would therefore potentially

have health benefits similar to that of dietary fibres in general. Improved

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laxation was observed in 14 subjects given 25 g RS (PROMITORTM) or 25 g

wheat bran fibre per day for 14 days in a randomized crossover design (Maki

et al., 2009). Both the RS and wheat bran increased faecal weight significantly

and did not differ from each other; both increased daily faecal weight above

150 g. This suggests that colon diseases associated with lesser laxation,

including diverticulosis/diverticulitis and IBS of the constipation type, may

be benefited by intake of RS. Colon cancer might also be more likely to be

prevented by RS intakes great enough to enhance laxation, based on the

findings of (Birkett et al., 1997).

15.3.2 RS, IBS and diverticulosis

No human studies have been performed on the role of RS in IBS to date, and

there are no clearly relevant animal models of this condition; neither has the

relation between RS intake and diverticulosis been studied. In an 18-month

study of nine groups of a total of 1800 Wistar rats given 0–17% wheat fibre,

incidence of diverticula was negatively associated with fibre intake and ranged

from 9% in rats fed the greatest fibre to 47% in rats fed no fibre. However, the

fibre intakes of the six treatment groups between 0–17% dietary fibre ranged

only from 0.5–4.2%, with incidence of diverticula ranging from 28–41%

(Fisher et al., 1985).

Estimated dietary fibre intakes in the US from the Continuing Survey of

Food Intakes of Individuals (1998), 10th–90th percentiles of intake ranged

from 7.1–24.8 g/d (Food&Nutrition Board, 2005) or�1.4–5.0% by weight of

diet (based on 500 g intake). This would suggest modest variance across

dietary fibre intakes in the US population in incidence of diverticula, which is

a common condition associated with aging. The significance of RS as a type of

dietary fibre in preventing diverticula in humans is probably scant.

15.3.3 RS and IBD

The ability of RS to prevent or reduce inflammatory bowel diseases (IBD –

e.g. ulcerative colitis) has been better studied and seems promising. In

Sprague-Dawley rats induced with colitis by 5% dextran sulphate sodium

in drinking water given for seven days, subsequent intake of 4% RS (type 3)

for seven days significantly improved colon histopathology scores compared

to controls, and also increased SCFA concentrations in cecal contents (Moreau

et al., 2003).

In rats with colitis induced by trinitrobenzene sulphonate (TNBS), fed

�6% RS for 14 days before and 21 days after TNBS, increased SCFA

concentrations were found in caecal and colon contents and healing was


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accelerated compared with controls (Jacobasch et al., 1999). Pigs fed 17–24%

RS (raw potato starch, type 2 RS), compared with �5–7% RS from corn

starch, for 14 weeks, showed increased butyrate in colon contents and reduced

immune system reactivity in intestinal epithelia, including reduced numbers

of T-helper cells. This suggests that pigs fedmore RSwould be able to respond

to intestinal inflammation better (Nofrarias et al., 2007).

Twenty-two patients with quiescent ulcerative colitis (UC) consumed 60 g

oat bran (20 g dietary fibre) for 12 weeks. Faecal butyratewas increased at four

weeks, compared with baseline, but not at later time points in these subjects.

They did not show an increase in gastrointestinal symptoms during this time,

compared with ten control UC patients not ingesting oat bran, who showed

increased symptom scores according to a questionnaire (Hallert et al., 2003).

This study suggests that RS, which would share fermentation characteristics

with oat bran, might also be of some benefit in ulcerative colitis. However,

well-controlled trials of RS in colitis patients remain to be reported. Such

studies would be quite difficult to do; recruitment of sufficient subjects of

similar disease state, measuring compliance with test diets and monitoring of

treatments and symptoms over sufficient time periods would be challenging.

The needs of patients for standard treatments, depending on disease state,

might confound effects of RS.

15.3.4 RS and colon cancer risk – human studies

As with IBD, the role of RS in colon cancer risk has been studied to a very

limited extent. The following review of existing data generally does not

support the ability of increased RS intake to decrease colorectal cancer risk in

humans. This review updates similar findings (Young, 2004). Faecal samples

from 17 Native Africans (from South Africa), 17 African Americans and

18 Caucasian Americans showed that the Africans had greater faecal SCFA

concentrations than either group of Americans. This finding was attributed to

the diet of the Africans, assumed to consist largely of maize porridge, which

could contribute 30–60 g of RS per day (O’Keefe et al., 2009).

Subjects who had been treated previously for hereditary nonpolyposis

colon cancer (Lynch syndrome) were given 13 g RS/d (Novelose) for 29

months or a placebo (waxy maize starch, composed entirely of amylopectin).

The incidence of colon neoplasms after this time period did not differ between

the two treatments; 18.7% of 358 subjects given RS vs. 18.4% of 369 subjects

given the placebo (Burn et al., 2008).

Twenty-four pre-operative colorectal cancer patients given the same RS as

in the Burn et al. study showed a lesser proportion of mitotic cells in the upper

half of colon crypts than did ten similar subjects given amylopectin placebos


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after ingesting the starches for four weeks (Dronamraju et al., 2009). The

reduction in this marker of premalignancy indicated that RS may protect

against colon cancer. In sporadic adenoma patients, 19 g of RS consumed

daily for two months did not alter epithelial cell proliferation in colorectal

biopsies (bromodeoxyuridine labelling index) compared with controls

ingesting placebo starch (n¼ 28 per treatment; van Gorkom et al., 2002).

After colon adenomectomy, 23 patients consumed 45 g of highly digestible

maltodextrin or digestion resistant maltodextrin (28 g/d of RS type 2) for four

weeks. Faecal bile acid concentrations decreased significantly in subjects

given RS, but SCFAs and colorectal cell proliferation did not differ between

the treatments (Grubben et al., 2001).

To date, few studies have been done on RS and colon cancer risk in humans.

Two studies suggest possible antineoplastic effects of RS, in suppressing

colon cell proliferation (Dronamraju et al., 2009) and lessening faecal bile

acids (Grubben et al., 2001) which may be colon tumour promoters (Nagen-

gast et al., 1995). Three studies (van Gorkom et al., 2002; Grubben et al.,

2001; Burn et al., 2008) did not show differences in colon cell proliferation

between RS and placebo treatments in colon cancer patients. Such patients are

at greater risk of colon cancer than the general population and are considered

good populations to attempt dietary interventions. Because cancers develop

over the lifespan and more often emerge later in life, long-term dietary

intervention trials of RS in general populations or in individuals with genetic

traits predisposing to colon cancer might be considered, if animal models are

sufficiently supportive of a protective role for RS.

15.4 COLON CANCER PREVENTION – ANIMALMODELS

There has been considerable interest in the ability of RS to reduce the yield

of colon preneoplastic lesions and colon cancers in rodent models. In most

cases, RS type 1 or 2 has been fed in diets preceding and/or following treatment

with a chemical carcinogen. In one study, Sprague-Dawley rats were fed diets

containing 0%, 10% or 20% raw high-amylose corn starch for four weeks prior

to treatment with azoxymethane (AOM), and colon cancers were assessed 25

weeks after AOM treatment. Both doses of starch inhibited colon tumour

development and increased SCFA including butyrate in the distal colon (Le Leu

et al., 2007).

In a series of studies, the ability of RS to counteract DNA damage in the

colon that was induced by high protein intakes was assessed. A high-amylose

butrylated starch was compared with high-amylose starch in preventing the


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DNA damage caused by a high protein diet, as measured by the comet assay in

rats. Both diets were effective in protecting against DNA damage by the high

protein diet, but the diet with butrylated starch was the most effective. This

protection paralleled increasing butyrate in the large intestine (Bajka et al.,

2008). Further, feeding 20% high-amylose corn starch for four weeks with

cooked beef or chicken prevented the genetic damage caused by these meats

(Toden et al., 2007).

Although much of the research has focused on RS type 1 or 2, one study

was conducted with a hydrothermally processed type 3 resistant starch

(Novelose 330) fed for 20 weeks following treatment of Sprague-Dawley

rats with 1,2-dimethylhydrazine. The RS completely prevented the develop-

ment of tumours, compared to rats fed control starch. The RS-3 starch also

increased apoptosis and decreased proliferation in the colon (Bauer-Mari-

novic et al., 2006).

Since not all studies have observed reduced tumour yields in rodents fed

RS, a study was designed to assess the timing of feeding RS relative to AOM

treatment in Wistar rats. Raw potato starch was fed for a three-week interval,

either before or after AOM treatment, and the number of aberrant crypt foci

(ACF) was increased in the rats fed RS before AOM, in comparison with those

fed control diet, while the yield of ACF was suppressed in a dose-dependent

manner in the rats fed RS following AOM treatment (Liu & Xu, 2008). These

authors concluded that RS fed following carcinogen treatment was more

likely to inhibit the colon lesions induced by AOM than RS fed preceding

carcinogen treatment.

SCFA, including butyrate, acetate and propionate, have been implicated as

mediators of the resistant starch impact on colon carcinogenesis (Topping &

Clifton, 2001). These short-chain fatty acids are produced by microbes that

ferment the resistant starch that reaches the large intestine. The hypothesis

generally implicates production of butyrate, because of observations that

butyrate promotes differentiation of colonocytes, but data in support of this

hypothesis have been mixed. For example, studies comparing cellulose and

high-amylose resistant starch revealed the greatest increase in caecal butyrate

with the resistant starch, but the greatest inhibition of colon tumours with

cellulose (Sakamoto et al., 1996). Furthermore, Nakanishi et al. (2003) treated

rats with Clostridium butyricum, and with or without high-amylose starch,

during treatments with AOM. Treatment with C. butyricum alone increased

butyrate in the caecum, but it did not decrease aberrant crypt foci. However,

treatment with both C. butyricum and high-amylose starch decreased aberrant

crypt foci and increased acetate and propionate concentrations in the caecum.

Elevatedb-glucuronidase activitywas also observed in the caecal contents fromthe rats treated with both C. butyricum and high-amylose starch.


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Studies were conducted with AOM-treated F344 rats, and the key end-

points were ACF and mucin-depleted foci (MDF). Secondary endpoints were

caecal pH, weight and SCFA concentrations (Zhao et al., 2011). ACF are

morphologically altered crypts, alone or in cluster, identified by microscopic

examination of methylene blue-stained whole mount preparations of colonic

mucosa from carcinogen-treated rodents (Bird, 1995). They are characterized

by being at least two to three times larger than normal crypts, having increased

pericryptal space and a slit-like opening, with thicker epithelium that stains

darker with methylene blue.

In recent years, a subset of AOM-induced ACF were believed to be better

predictors of cancer; these are MDF that are observed in alcian blue-stained

tissue which stains mucins. MDF are identified by the absence of the deep blue

staining indicative of mucins. These lesions highly correlated to tumour

development in AOM-treated rats (Caderni et al., 2003). More recently, these

lesions were observed in humans (Femia et al., 2008). AOM is selective for the

colon, and only a few small bowel/stomach tumours are noted. Carcinomas are

generally found approximately 30 weeks after carcinogen administration, and

tumour incidence and multiplicity can be accurately titrated (Pories et al.,

1993). AOMproduces both adenomas and carcinomas, with a predominance of

lesions induced in the distal colon, consistent with human sporadic cancers.

A high-amylose starch with exceptionally high resistance to digestion

was prepared by complexing high-amylose starch with lipids in a manner

that was expected to make it less digestible and, thus, to pass into the large

intestine and modify the gut microbial populations (Hasjim et al., 2010).

Studies evaluated the impact on pre-cancer lesions in the colon of rats by

including this resistant starch, fed in comparison with raw high-amylose

corn starch or normal corn starch, or cooked with high moisture as would be

used in a pudding, in comparison with similarly cooked high-amylose corn

starch or normal corn starch. The rats were fed a normal corn starch, a high-

amylose starch or the processed high-amylose starch after treatment with the

carcinogen AOM (Zhao et al., 2011).

This study showed that the high-amylose starch and the processed high-

amylose starch reduced pH and increased caecum weight, irrespective of

whether they were cooked by a high-moisture method. The raw high-amylose

starch reduced ACF, but ACF were not reduced by the processed starch in

comparison with raw normal starch or raw high-amylose starch (Zhao et al.,

2011). When all three starches were cooked by boiling in water, the processed

starch reduced MDF the most, high-amylose starch had an intermediate

reduction and the most MDF were found in the rats fed the normal starch.

Parallel observations were made with ACF, the more abundant precancerous

lesion induced by AOM (Zhao et al., 2011). This research supports the ability


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of the processed high-amylose starch to prevent the development of colon

cancer in this rat model.

15.5 CONCLUSIONS

Research in humans and laboratory animals suggest potential health benefits

of dietary RS in protection against diseases of the colon, including irritable

bowel syndrome, inflammatory bowel diseases (colitis) and colon cancer. To

date, the research suggests that the type and cooking of RS, the control starch

used, the time of feeding relative to the disease process and the nature of the

disease may all impact the ability of RS to benefit colon health. Although the

research has shown considerable promise for RS, further research is needed at

all levels to provide a firm foundation for the use of RS in the maintenance of

human health.

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Index

Note: Page numbers in italic refer to figures and tables.

acetate 231, 232, 234, 240, 242,

273

ActistarTM 102

acylated starches 55–6, 63–4

adipose fat 241–2, 243

ae starch see amylose-extender

AGPase 5, 6

All-Bran1 201

amylases

a-amylase 4, 96–7, 115–16

b-amylase 3, 4

isoamylase 83

natural inhibitors 120

resistance to 47–50, 103–5

see also enzymatic hydrolysis

amylopectin 1–2, 102

amylose/amylopectin ratios 3–4

helices 98

retrogradation and digestion

rate 118–19

types and granule structure 24–6

and waxy starch 8–9

amylopectin-lipid complex 79, 85–6

amylose

allomorphs/structure 1, 102

amylopectin/amylose ratios 3–4

helices 98

starch biosynthesis and 5, 8, 11–12,

14

see also high-amylose starch

amylose-extender starch

flour replacement product 168

role of HAM gene 36–7

structures 26–7

amylose-lipid complex (RS5) 79–80,

91–2, 122–3

advantages of 80

enzyme resistance and 80–1

complex crystallinity 82

granular starch properties 82

lipid structure 81

formation of starch-lipid

complex 83

debranching and RS content

85–6

fatty-acid choice and RS

content 83–5

health benefits of 87–91

products and applications 86–7

AOAC methodology 133–6, 137, 138,

146, 147

appetite and satiety 211, 215

appetite regulation 215–16, 239–40,

241, 242




279

BINDEX 07/22/2013 7:47:26 Page 280

appetite and satiety (Continued)

anorexigenic and orexigenic

hormones 216

role of hypothalamus 215–16

measuring effects of RS intake

human data 216–17, 221–5,

239–40

rodent data 218–21

proposed mechanisms for RS

effects 217–18, 229

satiety, satiation and fat

deposition 239–42

applications see food and beverage

applications

assay see measurement

bacteria see fermentation; microbiota

Bacteroidetes bacteria 252–3, 255,

257–8, 261

baking see biscuit baking

barley mutants 6, 12

batters 67

beta-cell function 199–202

and glucose toxicity 199–200

and serum free fatty acids 200

Bifidobacterium spp. 253, 255, 258,

269

biosynthesis, starch

overview of 4–6, 13–14

starch branching enzymes 11–12

starch debranching enzymes 13

starch synthases 6–11

biscuit baking 167

ae flour replacement starch

(RS3) 168–72

cookies 172–5

crackers 175–8

extruded ready-to-eat cereals

178–89

Brittle-2 mutant 6

bowel diseases see colon health

branching enzymes 11–12

breads 65, 66, 86

butyrate 230–1, 233, 234, 243, 258

and colon health studies 27, 268–9,

272

butyrylated starch 63–4

cancer, colon 89–91, 231, 268, 271–2

animal models and prevention

272–5

cereal starches

compared to tuber starches 3, 48

mutants 6, 7, 10–12

see also high-amylose starch

chemically modified (RS4) starch 46,

103, 105, 132, 145

amylase resistance 47–9, 103–4

mechanism 49–50

cross-linked starches 50–4

physicochemical properties 57–9

physiological responses to 60–4

food grade starches 57–8, 105

performance in food products

65–8

health benefits 60–5

pyrodextrinized starches 56–7

physicochemical properties 59


substituted starches 54–6


chloroplasts 4–5

cholesterol 237–8

citrates, starch 53–4, 59–60

colon health studies 267–8

cancer 268, 271–2

animal studies 272–5

inflammatory bowel diseases 268,

270–1

irritable bowel syndrome 268, 270

related to microbial

fermentation 268–9

colonic fermentation/microbiota see

fermentation; microbiota

constipation 267–8, 269–70

cookies 66

baking 172–5

cooking 4, 26

280 Index

BINDEX 07/22/2013 7:47:26 Page 281

high-amylose maize (RS2)

starches 27

in presence of lipids 80–1

see also biscuit baking

corn see maize

crackers see biscuit baking

cross-linked (RS4) starches 50–4, 105,

145–6

in vitro enzymatic testing see wheat

starch



crystalline starch 28–9, 99

debranching enzymes 5, 13, 83

dextrins, resistant 56–7, 59, 104, 105,

240

in vitro testing of wheat starch

148–9, 151–2, 153

diabetes 43, 95, 208

carbohydrate quality/quantity and

risk 193–5

complications and prevalence

192–3

definition and types 191–2

insulin sensitivity and

secretion 195–6, 209, 210,

235–6

glycemic impact of

carbohydrate 197–8

low-GI foods and improved beta-cell

function 199–200

see also glycemic response

diet, healthy 208

dietary fat see fat

dietary fibre 43, 44, 45, 46, 57–8, 105

definitions of 13, 136, 137, 138

measurement of 131, 138–42

digestion, starch 2–3, 23–4, 96–7,

115

chronology of indigestibility

studies 44–7

digestibility of

biosynthetic mutants 7

cross-linked wheat starch 149,

160–2

factors affecting 3–4

slowly versus readily digestible

starch 113–15

see also enzymatic hydrolysis

diverticulosis 268, 270

enzymatic hydrolysis 2, 3, 24, 96–7,

115–16

amylose-lipid complex (RS5) 79,

80–1

factors affecting 97–100

high-amylose starches 27

inhibitors of 120

resistance to

and molecular structure 102–5

and physical structure 97–102

enzymatic testing see measurement

Eubacterium spp. 256

extruded cereal 178–89

fat 208

deposition 241–2, 243

lipid metabolism 236–8

fatty acids see short-chain fatty acids

fermentation, microbiotic 209–10,

212, 217–18, 220, 221, 260

metabolic studies reviewed 230–5,

239–40

colon health 268–9

microbiology of see microbiota

fiber/fibre see dietary fibre

Fibersol 105

Fibersym1 179, 183–9

Firmicutes bacteria 252–3, 254, 257

flour replacement (RS3) starch 168

food and beverage applications

amylose-lipid complex (RS5) 86–7

high-amylose (RS3) starch 168–72

cookies 172–5

crackers 175–9

extruded cereal 179–89

modified (RS4) starches 65–8

Index 281

BINDEX 07/22/2013 7:47:26 Page 282

food grade starches 57–8, 105

food viscosity 120

gelatinization 4, 26

cross-linked wheat starch 150,

152–3

high-amylose starch 29, 30, 35,

168

and slowly digestible starch 118–19

see also retrograded starch

GI concept 111–12

GLP-1 (glucagon-like peptide or

polypeptide) 201–2, 238–9,

243

role in appetite 216–25, 241

glucan polymers 1–2

and starch biosynthesis 5

glucose 2, 112–13

slowly available concept 116

toxicity/tolerance 199–200, 235–6

see also glycemic response

a-glucosidases 96–7, 115–16

natural inhibitors 120

glutarates, starch 54, 60

glycemic index (GI) 111–12, 113,

194–5, 197–9

low-GI foods and beta-cell

function 199–200

glycemic load (GL) 194–5, 235, 243

glycemic response 2–3, 60–1, 112

amylose-lipid complex (RS5) 87–9

low GI carbohydrates 197–9

natural inhibitors and food

viscosity 120

regulation by resistant starch

208–11, 212, 235–6

slowly versus readily digestible

starch 113–15

granule see starch granule

HAM gene 36–7

health benefits 2–3, 43, 60–5

amylose-lipid complex (RS5)

87–91

healthy diet 208

see also colon health

helices, starch 98

Hi-MaizeTM 179, 183–9, 238

high-amylose maize (RS2) starch

11–12, 14, 23–7, 101

amylose-extender mutant

role of HAM gene 36–7

structures 26–7

elongated starch granules 26–7

formation in the amyloplasts

33–5

structures 27, 31–3, 34

resistant starch

formation and kernel

development 29–31

location in the starch granule

35–6

semi-crystalline amylose/IC

structures 28–9

use in amylose-lipid complexes 83,

84, 85

use in (RS3) flour replacer 168

high-amylose modifier (HAM)

gene 36–7

hunger see appetite

hydroxypropylation 54–5

hypoglycemia see glycemic control

hypothalamus 215, 220, 242

insulin

sensitivity and resistance to 195–7,

209, 210, 235–6, 241

see also diabetes; glycemic

response

irritable bowel syndrome 268,

270

isoamylase 83

Lachnospiraceae bacteria 256

laxation 267–8, 269–70

lipid complexes see amylose-lipid

complex


282 Index

BINDEX 07/22/2013 7:47:26 Page 283

maize starch 12–13, 23

mutant starches 6, 7, 10, 11–12, 14,

23

structure 26–7

see also high-amylose maize (RS2)

starch

maltase-glucoamylase 96–7, 115–16

maltodextrins 64, 65, 105

measurement of resistant starch

131–3

AOAC official method 133–6,

146

enzymatic of wheat starch see wheat

starch

in vitro and in vivo compared 146–8

integrated procedure for total dietary

fibre 136–42

metabolic studies, review of

fermentation and colonic

metabolism 230–5, 239,

259–62

GIP, GLP-1 and PYY

secretion 238–9

glycemic response and glucose

tolerance 235–6


satiety and fat deposition 239–42

see also appetite and satiety

metagenomics 256–7

microbiota, gut 60, 113, 229, 239,

251–2

colon health studies 268–9

common microbes 252–3

impact of RS 259–62

microbiological studies 260–2

16S rRNA-based studies 253–4

cultural studies 252–3

metagenomics and 256–7

starch as a growth substrate 257–8

metabolite cross-feeding 258

starch utilization systems 254–5

Bacteroides spp. 255

Bifidobacterium spp. 255–6

Lachnospiraceae 256

Ruminococcaceae 256

see also fermentation

modified starch see chemically modified

molecular structure, starch 102–3, 104

mutant starches 6, 7, 10, 11–12, 14, 23

see also high-amylose maize (RS2)

starch

Nutriose1 105

obesity 43, 95, 193, 207

fat deposition studies 241–2

pasta 66–7, 67–8

peptide YY see PYY

phosphated distarch phosphate 57

phosphorylated starches 47, 48,

50–3

in vitro testing see wheat starch



photosynthesis 4–5

products see food and beverage

applications

PromitorTM 105, 235, 240

propionate 231, 233, 234, 242, 243,

273

pullulanase 83

pyrodextrinized (RS4) starches 56–7,

59


physiological responses to 64, 269

PYY (peptide YY) 216, 238–9, 243

role in appetite 217–24, 241

readily digestible starch (RDS) 2–3

glycemic response compared to

SDS 113–15

resistant starch (RS), overview of

definitions 2–3, 44, 229

factors affecting enzyme

resistance 97–105

history of indigestibility studies

44–7

Index 283

BINDEX 07/22/2013 7:47:26 Page 284

resistant starch (RS), overview of

(Continued)

measurement of see measurement

physiological effects and benefits

2–3, 60–5, 207, 259–62

types and classification see RS1/2/3/

4/5

retrograded starch 3, 44, 45, 47, 80,

101, 132

Roseburia spp. 256

RS1/2/3/4/5 starches

described 3, 44, 45, 46, 47, 80,

100–2, 167–8

RS2 starch see high-amylose maize

(RS2) starch

RS3 starch 101–2, 167–72

RS4 starch see chemically modified

RS5 starch see amylose-lipid

complex

Ruminococcaceae bacteria 256

satiety see appetite and satiety

SDS see slowly digestible starch

second-meal effect 121, 210, 235

short-chain fatty acids 60, 62, 63,

209–10

and amylose-lipid complex 84

and beta-cell function 200

and colon health 268, 272, 273

fermentation in the gut 209–10,

212, 217–18, 220, 221

studies reviewed 230–5, 242

Shrunken-2 mutant 6

slowly digestible starch (SDS) 2–3,

111–13, 132

basic concept of SDS 111–13,

116–17

digestion of starch 115–16

glycemic response compared to

RDS 113–15

physiological/structural

factors 116–17

food matrix structures 117–18

food transit time 121

starch structures 118–19

viscosity and enzyme

inhibitors 120–1

strategies to make SDS 121–3

sodium trimetaphosphate 50, 51–2

starch, general overview of

classification/types 2–3

composition and structure 1–2,

23–6, 27, 96, 97–8

crystalline structures 99

granules see granule structure

molecular structure 102–3

starch helices 98

see also resistant starch

starch citrates 53–4, 59–60

products 67–8

starch digestion see digestion

starch glutarates 54, 60

starch granule structures 25, 26, 27,

99–100

cross-linked wheat starch 153–60

elongated high-amylose 27, 31–3,

34

and lipids 82

and resistant starch location 35–6

starch synthases 5, 6–8

synthase-I 8, 9

synthase-II 9–10

synthase-III 10–11

synthase-IV 11

starch types 2–3

ae corn starch III RS (X150) 168

see also RS1/2/3/4/5

structures, starch see starch; starch

granules

substituted (RS4) starches 54–6, 56–7

sucrase-isomaltase 96, 97, 115

sugary-1 mutants 13, 23

synthases see starch synthases

tortillas 66

total dietary fibre, assay of 136–42

transit time 115, 121

triacylglycerols (triglycerides) 373–4

284 Index

BINDEX 07/22/2013 7:47:26 Page 285

tuber starches 3, 48

Type 2 diabetes see diabetes

types of starch see starch types

viscosity, food 120

wafers 67

waxy starches 8–9, 54–5, 56, 80, 86

wheat starch (cross-linked), in vitro

testing of 145–8

materials and methods 148–50

results and discussion 151–63

Index 285

Documents

Resistant Starch: Sources, Applications and Health … · Press The discovery of resistant starch represents one of the major developments in our understanding of the importance of